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Methods in
Molecular Biology 1936
David A. Lyons
Linde Kegel Editors
Oligodendro-
cytes
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Oligodendrocytes
Methods and Protocols
Edited by
David A. Lyons and Linde Kegel

Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK
Editors
David A. Lyons Linde Kegel
Centre for Discovery Brain Sciences Centre for Discovery Brain Sciences
University of Edinburgh University of Edinburgh
Edinburgh, UK Edinburgh, UK
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-9070-2 ISBN 978-1-4939-9072-6 (eBook)
https://doi.org/10.1007/978-1-4939-9072-6
Library of Congress Control Number: 2019930142
© Springer Science+Business Media, LLC, part of Springer Nature 2019

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Cover Caption: Single oligodendrocyte, labelled with GFP in a transgenic zebrafish. Image courtesy of Dr. Rafael
Almeida, Lyons Lab, University of Edinburgh, Edinburgh, UK.
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Preface
We have known for nearly a century that the oligodendrocyte is the glial cell of the central
nervous system that generates myelin. Similarly, we have known for decades that the
presence of myelin on axons speeds up conduction velocities, by virtue of the fact that
myelin sequesters key ion channels to the short nodes of Ranvier between consecutive
myelin sheaths and due to the remarkable properties of the lipid-rich myelin sheath itself.
The myelination of axons allows rapid information propagation throughout specific neural
circuits, and as a consequence disruption to myelin has been known to underpin diseases of
the central nervous system, such as multiple sclerosis, for a very long time.
However, it was not until the expansion of a myriad of advanced, molecular, cellular, and
imaging techniques over the past two decades that our knowledge of how oligodendrocytes
develop, mature, and make myelin has started to come together. We now have a very good
understanding of the molecular genetic mechanisms that specify oligodendrocyte progeni-
tor cells in distinct domains of the developing nervous system, and we are starting to
understand how these cells migrate throughout the CNS. Indeed, it is now clear that
oligodendrocyte progenitor cells reside throughout the gray and white matter of our CNS
lifelong, and that they retain a capacity to generate mature oligodendrocytes, both in the
healthy nervous system and in response to damage or loss of myelin in diseases, such as MS.
Our understanding of oligodendrocyte differentiation is now also being elucidated
through complementary in vitro and in vivo molecular and cellular analyses. In addition,
work over the past decade or so has also started to elucidate how oligodendrocytes subse-
quently select which axons to myelinate and how myelin sheath growth and remodeling
occurs thereafter. An area of investigation that has undergone something of a renaissance in
recent years is based around the fact that many stages of oligodendrocyte progenitor cell
development, oligodendrocyte differentiation, and myelination are responsive to changes in
neuronal activity. In-depth analyses of how neuronal activity regulates oligodendrocytes
have been facilitated by numerous new techniques and have led to the concept that dynamic
regulation of myelination may occur lifelong and may represent a form of functional nervous
system plasticity. This concept is supported by reductionist molecular, cellular, and electro-
physiological studies noted in this volume and by parallel system-level imaging of animal
models and humans, which is also discussed within this volume.
Reflecting the idea that oligodendrocytes remain responsive to and active regulators of
neural circuit function throughout life is the fact that new functions for oligodendrocytes
and myelin have recently emerged, highlighting the fact that this cell lineage does more than
converge on producing a static insulator to facilitate conduction. Oligodendrocytes regulate
ion homeostasis and thus neuronal excitability; they provide metabolic support to axons and
likely have an array of functions that we will begin to be able to unravel due to the
approaches outlined in this volume and others. The prediction that oligodendrocytes play
yet more functional roles than we currently understand is based on the fact that their
disruption is now implicated in many, if not most, diseases of the nervous system, from
neurodevelopmental disorders including autism, neuropsychiatric disorders including
schizophrenia, neurodegenerative disorders including Alzheimer’s disease, motor neuron
disease, and others. In addition, oligodendrocytes and myelin exhibit dynamic changes
v
vi Preface
across the life course that tightly correlate with the emergence, stability, and decline of
cognitive performance.
In this volume, we bring together a series of methods that bridge in vitro and in vivo
approaches, model organisms, analyses of health and disease, and scales from molecular
through to system. We take a bottom-up approach and start with fundamental molecular
analyses of oligodendrocytes and myelin and move through an array of in vitro and ex vivo
molecular-cellular-electrophysiology-based approaches from mouse to man. In the second
part of the volume, we collate methodologies for in vivo analyses of oligodendrocyte
formation, homeostasis, and disruption, in zebrafish, Xenopus, mouse, and man. Additional
methods of relevance not included in this volume are brought together in a separate volume
on myelin, which we direct interested readers to.
We hope that this volume highlights the fast-paced and broad nature of research into the
formation, health, and function of the oligodendrocyte and serves as a useful reference
manual for the field.
Edinburgh, UK David A. Lyons

Linde Kegel
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Single-Cell RNA Sequencing of Oligodendrocyte Lineage
Cells from the Mouse Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sueli Marques, David van Bruggen, and Gonçalo Castelo-Branco
2 Isolation and Preparation of Cells from Focal Remyelinating
Central Nervous System Lesions for RNA Sequencing. . . . . . . . . . . . . . . . . . . . . . . 23
Claire L. Davies, Stefka Gyoneva, Anne Cotleur,
Richard M. Ransohoff, and Veronique E. Miron
3 Myelin: Methods for Purification and Proteome Analysis . . . . . . . . . . . . . . . . . . . . 37
Michelle S. Erwig, Dörte Hesse, Ramona B. Jung,
Marina Uecker, Kathrin Kusch, Stefan Tenzer, Olaf Jahn,
and Hauke B. Werner
4 In Vitro Generation and Electrophysiological Characterization
of OPCs and Oligodendrocytes from Human Pluripotent Stem Cells . . . . . . . . . 65
Dario Magnani, Siddharthan Chandran, David J. A. Wyllie,
and Matthew R. Livesey
5 Isolation and Culture of Oligodendrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Marie-Theres Weil, Giselheid Schulz-Ëberlin, Chaitali Mukherjee,
Wen Ping Kuo-Elsner, Isabelle Sch€ a fer, Christina Müller,
and Mikael Simons
6 A Neuron-Free Microfiber Assay to Assess Myelin Sheath Formation . . . . . . . . . . 97
Marie E. Bechler
7 Oligodendrocyte–Neuron Myelinating Coculture . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Matthew Swire and Charles ffrench-Constant
8 Teasing of Ventral Spinal Cord White Matter Fibers for the Analysis
of Central Nervous System Nodes of Ranvier. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Andrew A. Jarjour and Diane L. Sherman
9 Whole-Cell Patch Clamp Recordings from Oligodendrocyte
Lineage Cells in Brain Slices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Sylvia Agathou and Ragnhildur Thora Kárado ttir
10 Ex Vivo Slice Cultures to Study Myelination, Demyelination,
and Remyelination in Mouse Brain and Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . 169
Sowmya Sekizar and Anna Williams
11 Forward Genetic Screen Using Zebrafish to Identify New Genes
Involved in Myelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Linde Kegel, Maria Rubio, Rafael G. Almeida, Silvia Benito,
Anna Klingseisen, and David A. Lyons
12 Manipulating Neuronal Activity in the Developing Zebrafish
Spinal Cord to Investigate Adaptive Myelination. . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Jill M. Williamson, David A. Lyons, and Rafael G. Almeida
vii
viii Contents
13 A Drug-Inducible Transgenic Zebrafish Model for Myelinating

Glial Cell Ablation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Marja J. Karttunen and David A. Lyons
14 Conditional Demyelination and Remyelination in a Transgenic
Xenopus laevis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Abdelkrim Mannioui and Bernard Zalc
15 Conditional Mutagenesis in Oligodendrocyte Lineage Cells. . . . . . . . . . . . . . . . . . 249
Sandra Goebbels and Klaus-Armin Nave
16 Recent Advances in Live Imaging of Cells of the Oligodendrocyte
Lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Jaime Eugenin von Bernhardi and Leda Dimou
17 The DTA Mouse Model for Oligodendrocyte Ablation
and CNS Demyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Maria Traka
18 Human Glial Chimeric Mice to Define the Role of Glial
Pathology in Human Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
John N. Mariani, Lisa Zou, and Steven A. Goldman
19 A Method to Detect Cytochrome c Oxidase Activity
and Mitochondrial Proteins in Oligodendrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Graham R. Campbell and Don J. Mahad
20 Transmission Electron Microscopy of Oligodendrocytes and Myelin . . . . . . . . . . 343
Marie-Theres Weil, Torben Ruhwedel, Martin Meschkat,
Boguslawa Sadowski, and Wiebke Möbius
21 Toxin-Based Models to Investigate Demyelination and Remyelination . . . . . . . . 377
Christopher E. McMurran, Chao Zhao, and Robin J. M. Franklin
22 Magnetic Resonance Techniques for Imaging White Matter. . . . . . . . . . . . . . . . . . 397
Cassandra Sampaio-Baptista, Kata Diosi, and Heidi Johansen-Berg
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Contributors
SYLVIA AGATHOU Department of Veterinary Medicine, Wellcome Trust-Medical Research

Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
RAFAEL G. ALMEIDA Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
MARIE E. BECHLER MRC Centre for Regenerative Medicine, MS Society Edinburgh Centre
for MS Research, The University of Edinburgh, Edinburgh, UK
SILVIA BENITO Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
GRAHAM R. CAMPBELL Centre for Clinical Brain Sciences, University of Edinburgh,
Edinburgh, UK
GONÇALO CASTELO-BRANCO Laboratory of Molecular Neurobiology, Department of Medical
Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Ming Wai Lau
Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet, Stockholm, Sweden
SIDDHARTHAN CHANDRAN Centre for Clinical Brain Sciences, The University of Edinburgh,
Edinburgh, UK; Euan MacDonald Centre, The University of Edinburgh, Edinburgh, UK
ANNE COTLEUR Biogen, Cambridge, MA, USA
CLAIRE L. DAVIES Centre for Reproductive Health, The Queen’s Medical Research Institute,
The University of Edinburgh, Edinburgh, UK
LEDA DIMOU Molecular and Translational Neuroscience, Department of Neurology, Ulm
University, Ulm, Germany; Graduate School of Systemic Neurosciences, Ludwig-
Maximilians-University, Munich, Germany
KATA DIOSI NDCN Department, Wellcome Centre for Integrative Neuroimaging, FMRIB
Centre, University of Oxford, Oxford, UK
MICHELLE S. ERWIG Department of Neurogenetics, Max Planck Institute of Experimental
Medicine, Goettingen, Germany
JAIME EUGENIN VON BERNHARDI Molecular and Translational Neuroscience, Department of
Neurology, Ulm University, Ulm, Germany; Graduate School of Systemic Neurosciences,
Ludwig-Maximilians-University, Munich, Germany
CHARLES FFRENCH-CONSTANT MRC Centre for Regenerative Medicine, The Multiple
Sclerosis Research Centre, The University of Edinburgh, Edinburgh, UK
ROBIN J. M. FRANKLIN Wellcome Trust-MRC Cambridge Stem Cell Institute, University of
Cambridge, Cambridge, UK
SANDRA GOEBBELS Department of Neurogenetics, Max Planck Institute of Experimental
Medicine, Göttingen, Germany
STEVEN A. GOLDMAN Department of Neurology and the Center for Translational
Neuromedicine, University of Rochester Medical Center, Rochester, NY, USA; The
Neuroscience Center, Rigshospitalet, Copenhagen, Denmark; Center for Translational
Neuromedicine, Faculty of Health and Medical Sciences, University of Copenhagen,
Copenhagen, Denmark
STEFKA GYONEVA Biogen, Cambridge, MA, USA
DÖRTE HESSE Proteomics Group, Max Planck Institute of Experimental Medicine,
Goettingen, Germany
ix
x Contributors
OLAF JAHN Proteomics Group, Max Planck Institute of Experimental Medicine, Goettingen,
Germany
ANDREW A. JARJOUR MRC Centre for Regenerative Medicine and MS Society Edinburgh
Centre, Edinburgh bioQuarter, The University of Edinburgh, Edinburgh, UK
HEIDI JOHANSEN-BERG NDCN Department, Wellcome Centre for Integrative
Neuroimaging, FMRIB Centre, University of Oxford, Oxford, UK
RAMONA B. JUNG Department of Neurogenetics, Max Planck Institute of Experimental
RAGNHILDUR THÓRA KÁRADÓTTIR Department of Veterinary Medicine, Wellcome Trust-
Medical Research Council Cambridge Stem Cell Institute, University of Cambridge,
Cambridge, UK
MARJA J. KARTTUNEN Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
LINDE KEGEL Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh,
UK
ANNA KLINGSEISEN Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
WEN PING KUO-ELSNER Department of Biology, Molecular Cell Biology, Johannes
Gutenberg University, Mainz, Germany
KATHRIN KUSCH Department of Neurogenetics, Max Planck Institute of Experimental
MATTHEW R. LIVESEY Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
DAVID A. LYONS Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
DARIO MAGNANI Centre for Clinical Brain Sciences, The University of Edinburgh,
Edinburgh, UK; Euan MacDonald Centre, The University of Edinburgh, Edinburgh, UK
DON J. MAHAD Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh,
UK
ABDELKRIM MANNIOUI Sorbonne Universités UPMC, Univ Paris 06, Inserm, CNRS,
APHP, ICM-GH Pitié-Salpêtrière, Paris, France
JOHN N. MARIANI Department of Neurology and the Center for Translational
Neuromedicine, University of Rochester Medical Center, Rochester, NY, USA
SUELI MARQUES Laboratory of Molecular Neurobiology, Department of Medical
Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
CHRISTOPHER E. MCMURRAN Wellcome Trust-MRC Cambridge Stem Cell Institute,
University of Cambridge, Cambridge, UK
MARTIN MESCHKAT Max Planck Institute of Experimental Medicine, Department of
Neurogenetics, Electron Microscopy Core Unit, Göttingen, Germany
VERONIQUE E. MIRON Centre for Reproductive Health, The Queen’s Medical Research
Institute, The University of Edinburgh, Edinburgh, UK
WIEBKE MÖBIUS Max Planck Institute of Experimental Medicine, Department of
Neurogenetics, Electron Microscopy Core Unit, Goettingen, Germany; Center Nanoscale
Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany
CHRISTINA MÜLLER Institute of Physiology, University Medical Center, Johannes Gutenberg
University, Mainz, Germany
Contributors xi
CHAITALI MUKHERJEE Institute of Neuronal Cell Biology, Technical University Munich,

Munich, Germany; German Center for Neurodegenerative Disease (DZNE), Munich,
Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
KLAUS-ARMIN NAVE Department of Neurogenetics, Max Planck Institute of Experimental
Medicine, Göttingen, Germany
RICHARD M. RANSOHOFF Third Rock Ventures, Boston, MA, USA; Department of Cell
Biology, Harvard Medical School, Boston, MA, USA
MARIA RUBIO Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
TORBEN RUHWEDEL Max Planck Institute of Experimental Medicine, Department of
Neurogenetics, Electron Microscopy Core Unit, Göttingen, Germany
BOGUSLAWA SADOWSKI Max Planck Institute of Experimental Medicine, Department of
Neurogenetics, Electron Microscopy Core Unit, Göttingen, Germany; Center Nanoscale
Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany
CASSANDRA SAMPAIO-BAPTISTA NDCN Department, Wellcome Centre for Integrative
Neuroimaging, FMRIB Centre, University of Oxford, Oxford, UK
€
ISABELLE SCHAFER Institute of Physiology, University Medical Center, Johannes Gutenberg
GISELHEID SCHULZ-ËBERLIN Max Planck Institute of Experimental Medicine, Goettingen,
Germany
SOWMYA SEKIZAR MRC Centre for Regenerative Medicine and MS Society Edinburgh
DIANE L. SHERMAN Centre for Discovery Brain Sciences, The Chancellor’s Building,
Edinburgh bioQuarter, The University of Edinburgh, Edinburgh, UK
MIKAEL SIMONS Max Planck Institute of Experimental Medicine, Goettingen, Germany;
Institute of Neuronal Cell Biology, Technical University Munich, Munich, Germany;
German Center for Neurodegenerative Disease (DZNE), Munich, Germany; Munich
Cluster for Systems Neurology (SyNergy), Munich, Germany
MATTHEW SWIRE MRC Centre for Regenerative Medicine, The Multiple Sclerosis Research
Centre, The University of Edinburgh, Edinburgh, UK
STEFAN TENZER Institute of Immunology, University Medical Center, Johannes Gutenberg
MARIA TRAKA Department of Anatomy, College of Graduate Studies, Midwestern
University, Downers Grove, IL, USA
MARINA UECKER Proteomics Group, Max Planck Institute of Experimental Medicine,
Goettingen, Germany
DAVID VAN BRUGGEN Laboratory of Molecular Neurobiology, Department of Medical
Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
MARIE-THERES WEIL Max Planck Institute of Experimental Medicine, Department of
Neurogenetics, Electron Microscopy Core Unit, Göttingen, Germany; Center Nanoscale
Microscopy and Molecular Physiology of the brain (CNMPB), Göttingen, Germany; AbbVie
Deutschland GmbH and Co. KG, Ludwigshafen, Germany
HAUKE B. WERNER Department of Neurogenetics, Max Planck Institute of Experimental
ANNA WILLIAMS MRC Centre for Regenerative Medicine and MS Society Edinburgh
JILL M. WILLIAMSON Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
xii Contributors
DAVID J. A. WYLLIE Centre for Discovery Brain Sciences, The University of Edinburgh,
Edinburgh, UK
BERNARD ZALC Sorbonne Universités UPMC, Univ Paris 06, Inserm, CNRS, APHP,
ICM-GH Pitié-Salpêtrière, Paris, France
CHAO ZHAO Wellcome Trust-MRC Cambridge Stem Cell Institute, University of
Cambridge, Cambridge, UK
LISA ZOU Department of Neurology and the Center for Translational Neuromedicine,
University of Rochester Medical Center, Rochester, NY, USA
Chapter 1
Single-Cell RNA Sequencing of Oligodendrocyte Lineage

Cells from the Mouse Central Nervous System
Sueli Marques, David van Bruggen, and Gonçalo Castelo-Branco
Abstract
Single-cell RNA sequencing has emerged as a powerful technique for the identification of distinct cell
states/populations in complex tissues. We have recently used this technology to investigate heterogeneity of
cells of the oligodendrocyte lineage in the mouse central nervous system. In this chapter, we describe
methods to perform single-cell RNA sequencing on this glial cell lineage, and discuss experimental and
computational approaches to explore the potential and to tackle hurdles associated with this technology.
Key words Single-cell, RNA sequencing, Transcriptomics, Oligodendrocyte, Myelin, Central ner-
vous system
1 Introduction
The advent in the last decade of transcriptomic technologies, cou-

pling cDNA synthesis with next-generation sequencing (NGS), has
allowed the analysis of millions of RNA transcripts from distinct cell
populations and thus to determine key drivers in a plethora of
biological processes. The original RNA-sequencing protocols
were only compatible with pools of several thousand cells, resulting
in a loss of any detectable heterogeneity within the analyzed sample.
The first single-cell RNA sequencing (RNA-Seq) method was pub-
lished in 2009 elucidating the transcriptome of dissociated mouse
blastomeres [1]. Since then, microfluidic approaches such as the
Fluidigm C1 system have been developed, enabling cDNA synthe-
sis of hundreds of individual cells [2]. More recently, micro-droplet
approaches such as Drop-seq have been described, as well as com-
binatorial barcoding allowing processing of many thousands of cells
[3–5]. Recent studies have been focusing on cell lineage heteroge-
neity and deconstruction of tissues in populations and subpopula-
tions, revealing new biomarkers and leading to profound insights in
the cellular hierarchies existing within tissues [6–13]. We recently
David A. Lyons and Linde Kegel (eds.), Oligodendrocytes: Methods and Protocols, Methods in Molecular Biology, vol. 1936,
https://doi.org/10.1007/978-1-4939-9072-6_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019
1
2 Sueli Marques et al.
investigated heterogeneity within the oligodendrocyte lineage in

the mouse central nervous system in a similar manner [14, 15].
Oligodendrocytes (OLs) were first defined as an heterogeneous
cell population by Rio Hortega and Penfield in the 1920s
[16, 17]. Four types of oligodendrocytes were described, depend-
ing on the number of axons myelinated, the diameter of ensheathed
axons and their orientations. Subsequent studies further supported
and extended this view of morphological heterogeneity of oligoden-
drocyte populations in the central nervous system [18–23]. How-
ever, targeted ablation of oligodendrocyte precursor cell (OPC)
populations from specific ventral/dorsal origins in the forebrain
does not lead to behavior abnormalities in mice, with OPCs/oligo-
dendrocytes derived from non-ablated regions functionally com-
pensating the lost populations [24]. Furthermore, while
myelinating preferentially specific tracks, dorsal and ventral derived
oligodendrocytes present similar electrophysiological properties
[25]. In order to determine whether there is an intrinsic heteroge-
neity of cells of the oligodendrocyte lineage, we used single-cell
RNA sequencing with Fluidigm C1 technology to produce a com-
prehensive map of this lineage [6, 14]. Approximately 5000 cells
expressing OL lineage markers, from ten distinct regions of the
juvenile and adult mouse central nervous system (CNS), clustered
in 12 cell populations/states [14]. We found a relatively homoge-
neous OPC population that followed a narrow path of differentia-
tion through committed precursor cells (COP), newly formed OL
(NFOL 1 and 2), myelin-forming OL (MFOL 1 and 2) and diversi-
fying into 6 mature OLs (MOLs) (Fig. 1) [14]. The path of differ-
entiation was ubiquitous throughout the CNS, while the mature
OLs diversity was more region-specific, with distinct MOLs being
over-represented in specific areas. We also observed a segregation of
transcriptional profiles between four MOLs present in juvenile
(MOL1-4) compared to the two main MOLs enriched in adult
tissues (MOL5/6) [14]. The latter was enriched in genes involved
in synapse and neurotransmitter regulation [14], which could indi-
cate functional heterogeneity within the mature OLs. Using also
single-cell RNA-Seq, we recently found that progenitors of the
oligodendrocyte lineage transcriptionally converge during mouse
CNS development, to generate postnatal OPCs with similar gene
expression profiles regardless of the region of origin [15]. In this
book chapter, we describe a detailed protocol for single-cell RNA--
Seq of oligodendrocyte lineage cells in CNS tissues.
2 Materials
2.1 Tissue Collection 1. CNS tissue from wild-type mice or mice expressing eGFP
reporter or another fluorescent marker in cells belonging to
the oligodendrocyte lineage, such as Pdgfra-histone H2B-GFP
[26] or Pdgfra-Cre-RCE [27] mice.
Sequencing Oligodendrocytes 3
Fig. 1 Heatmap resulting from backspin algorithm of the 5072 cells from the OL lineage and showing markers
specific for the subclusters
2. Dissection tools.
3. Stereomicroscopes.
4. Vibratome VT1200S, Leica.
5. Syringe for perfusion.
6. Cutting solution—87 mM NaCl, 2.5 mM KCl, 1.25 mM
NaH2PO4, 26 mM NaHCO3, 75 mM Saccharose, 20 mM
Glucose, 1 mM CaCl2, and 2 mM MgSO4.
7. Oxygenation tank with 95% O2/CO2.
2.2 Tissue 1. Pasteur glass pipettes.

Dissociation 2. HBSS with calcium and magnesium (no phenol red)—Thermo
Fisher catalogue number: 14025050.
3. Neural tissue dissociation kit (P)—Miltenyi catalogue number:
130-092-628.
4. Adult brain dissociation kit, mouse and rat—Miltenyi cata-
logue number: 130-107-677.
5. Cell Trics® strainers 30 μm—Sysmex catalogue number:
25004-0042-2316.
2.3 FACS Sorting 1. FACS tubes—Corning, catalogue number #352058.

of OL 2. HBSS, no magnesium, no calcium, and no phenol red—
Thermo Fisher catalogue number: 14175095.
3. BSA 5%.
4. Nonsticky RNAse-free tubes—Thermo Fisher catalogue num-
ber: AM12450.
5. FACS machine (BD FACSAria III Cell Sorter B5/R3/V3
system).
2.4 Cell Capture 1. C1™ Single cell Reagent for mRNA seq—Fluidigm, catalogue
and Imaging number #100-6201.
2. C1 Single cell mRNA seq IFC microfluidic chip-sizes 5–10 μm
and 10–17 μm (catalogue numbers #100-5759 and #100-
5760, respectively).
3. Fluidigm C1 instrument.
4. Nikon TE2000E microscope.
5. μManager software.
2.5 Lysis, Reverse 1. Oligonucleotide sequences:

Transcription, and PCR
C1-P1-RNA- Bio-AAUGAUACGGCG All-RNA oligo
TSO ACCACCGAUNNNN
NGGG
C1-P1-T31 Bio-
AATGATACGGCGACC
ACCGATCGTTTTTTT
TTTTTTTTTTTTTT
TTTTTTTTTT
C1-P1-PCR-2 Bio-GAATGATACG
GCGACCACCGAT
C1-TN5-U PHO-CTGTCTCTTATA Mix with
CACATCTGACGC C1-TN5-x
to make adapter
2. Lysis buffer: 0.15% triton X-100, 1 U/μL Takara Rnase inhib-

itor, 4 μM reverse transcription primer C1-P1-T31, 5% C1
Loading Reagent, and 1:50,000 Life Technologies ERCC
Spike-In Mix 1.
3. Reverse transcription mix: 1 SuperScript II First-Strand
Buffer supplemented with 3 mM MgCl2, 1.5 mM dNTP,
4 mM DTT, 3.3% C1 Loading Reagent, 1.8 μM template-
switching oligo C1-P1-RNA-TSO, 1.5 U/μL TaKaRa RNase
inhibitor, and 18 U/μL Life Technologies Superscript II
reverse transcriptase.
4. PCR mix: 1.1 Clontech Advantage2 PCR buffer, 440 μM
dNTP, 530 nM PCR primer C1-P1-PCR-2, 5% C1 Loading
Reagent, and 2 Advantage2 Polymerase Mix.
5. C1 Harvesting Reagent.
6. Agilent Bioanalyzer.
7. Agilent High sensitivity DNA kit—catalogue number 5067-
4626.
2.6 Bioinformatic 1. MATLAB software—MathWorks.

Analysis, Quality 2. R statistical computing and graphic language—R Foundation.
Control, and Selection
2.7 Tagmentation 1. 96 different 10 transposome stocks (6.25 μM barcoded adap-

and Isolation of 50 tor C1-TN5-x, 40% glycerol, 6.25 μM recombinant Tn5 trans-
Fragments posase, where x denotes a well-specific barcode). Barcode
sequences can be found in Islam et al., 2014 [28].
2. Tagmentation buffer: 50 mM TAPS-NaOH, pH 8.5, 25 mM
MgCl2, and 50% DMF.
3. Dynabeads MyOne Streptavidin C1 beads.
4. BWT: 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 2 M NaCl,

0.02% Tween-20.
5. TNT: 20 mM Tris, pH 7.5, 50 mM NaCl, 0.02% Tween.
6. Qiagen Qiaquick PB.
7. Restriction mix: 1 NEB NEBuffer 4, 0.4 U/μL PvuI-HF
enzyme.
8. NaOH 100 mM.
9. HCl 100 mM.
10. Neutralization buffer: 200 mM Tris, pH 7.5, 0.05% Tween-20.
2.8 Illumina High- 1. KAPA library quantification kit—KAPA biosystems, catalogue

Throughput number KK4824.
Sequencing 2. Illumina HiSeq 2000 instrument.
3 Methods
Single-cell RNA-Seq has been shown to be a powerful tool to

profile cell-to-cell transcriptomic variability [6–13]. The general
workflow for single-cell RNA-Seq is common to many other tran-
scriptomic methods. First, a cell is lysed to obtain RNA, followed by
first strand synthesis by a dedicated reverse transcriptase, then a
PCR pre-amplification step is performed adding Illumina compati-
ble adapters. An important factor in single-cell RNA-Seq is the
manner (and efficiency) of single cell capture. Many existing disso-
ciation methods are tissue specific, and new protocols are constantly
being developed (see Note 1).
3.1 Tissue Collection Tissues were collected in oxygenated 1 cutting solution.

1. For embryonic stage, excise brain and spinal cord under stereo-
microscopes, after carefully removing meninges.
2. For postnatal and adult stages, intracardially perfuse mice with
oxygenated 1 cutting solution to clear the tissue. For dissec-
tion of specific regions, section brain or spinal cord in 300 μm
thick vibratome slices (kept in cold cutting solution with con-
stant oxygenation).
3.2 Tissue Depending on the age of the mice, different dissociation protocols
Dissociation to purify oligodendrocytes are used.
For embryonic stage, cut the tissue in pieces (<1 mm of thick-
ness) and perform manual dissociation using fire-polished Pasteur
pipettes with decreasing diameter until the suspension looks
homogeneous.
Myelin removal in postnatal and adult stages was required,
involving extra steps of purification after papain-driven tissue
dissociation, such as FACS sorting with strict gating parameters to

exclude myelin debris, or using specific gradients (see Note 2).
For postnatal day zero (P0) to P30 stages, Neural dissociation
kit with Papain from Miltenyi is used in a manual mode, adapting
manufacturer’s instructions:
1. Add 13.5 μL of 50 mM beta-mercaptoethanol to 10 mL of
Buffer X.
2. Prepare enzyme mixes:
Enzyme mix1—50 μL Enzyme P þ 1900 μL Buffer X
(complete).
Enzyme mix2—10 μL Enzyme A þ 20 μL Buffer Y.
Volumes given are for up to 400 mg of starting tissue material.
When working with less than 400 mg, use the same volumes as
indicated.
3. Fire-polish three glass Pasteur pipettes so that decreasing tip
diameters are achieved.
4. Preheat Enzyme mix1 at 37 C for 10–15 min.
5. Determine the weight of tissue in 1 mL of cold HBSS (w/o) to
make sure the 400 mg limit per digestion is not exceeded.
6. Cut brain into small pieces using a scalpel in a 35 mm diameter
petri dish.
7. Using a 1 mL pipette tip, add 1 mL of HBSS without ions
(w/o) and pipette pieces back into an appropriate-sized tube.
Rinse with HBSS (w/o).
8. Centrifuge at 300 g for 2 min at room temperature and
aspirate the supernatant carefully.
9. Add Enzyme mix 1.
10. Incubate in closed tubes for 15 min at 37 C, mixing gently
every 5 min.
11. Add the Enzyme mix2. Invert gently to mix.
12. Dissociate tissue mechanically using the wide-tipped, fire-
polished Pasteur pipette by pipetting up and down ten times
slowly. Avoid forming air bubbles.
13. Incubate at 37 C for 10 min, mixing gently every 5 min.
14. Dissociate tissue mechanically using the other two fire-
polished pipettes in decreasing diameter. Pipette slowly up
and down ten times with each pipette, or as long as tissue
pieces are still observable. Be careful to avoid the formation
of air bubbles.
16. Apply the cell suspension to a CellTric 30 μm, placed on a
15 mL tube.
17. Wash with 10 mL HBSS with ions.
18. Centrifuge cell suspension at 300 g for 10 min at room

temperature. Aspirate supernatant completely.
19. Resuspend in 1 mL cold cutting solution with 1% BSA.
20. Apply the cell suspension to another CellTric 30 μm, placed on
a FACS tube.
For adult stages, the Adult brain dissociation kit was used in a
manual mode, adapting manufacturer’s instructions (Miltenyi).
After dissociation was finished, a gradient was performed to clean
myelin, according to the kit’s protocol:
1. Prepare enzyme mixes:
Enzyme mix1—50 μL Enzyme P þ 1900 μL Buffer Z.
Enzyme mix2—10 μL Enzyme A þ 20 μL Buffer Y.
Volumes given below are for one adult mouse brain
(400–500 mg) in 1980 μL enzyme mix. When working with
less than 400 mg, use the same volumes as indicated.
2. Fire-polish three glass Pasteur pipettes so that decreasing tip
diameters are achieved.
3. Preheat Enzyme mix1 at 37 C for 10–15 min.
4. Determine the weight of tissue in 1 mL of cold HBSS (w/o) to
make sure the 400 mg limit per digestion is not exceeded.
5. Cut brain into small pieces using a scalpel in a 35 mm diameter
petri dish.
6. Using a 1 mL pipette tip, add 1 mL of HBSS without ions
(w/o) and pipette pieces back into an appropriate-sized tube.
Rinse with HBSS (w/o).
7. Centrifuge at 300 g for 2 min at room temperature and
aspirate the supernatant carefully.
8. Add Enzyme mix 1.
9. Incubate in closed tubes for 15 min at 37 C, mixing gently
every 5 min.
10. Add the Enzyme mix2. Invert gently to mix.
11. Dissociate tissue mechanically using the wide-tipped, fire-
polished Pasteur pipette by pipetting up and down ten times
slowly. Avoid forming air bubbles.
13. Dissociate tissue mechanically using the other two fire-
polished pipettes in decreasing diameter. Pipette slowly up
and down ten times with each pipette, or as long as tissue
pieces are still observable. Be careful to avoid the formation
of air bubbles.
15. Apply the cell suspension to a MACS SmartStrainer 70 μm,

placed on a 15 mL tube.
16. Apply 10 mL of cold PBS onto the strainer.
17. Centrifuge cell suspension at 300 g for 10 min at 4 C.
Aspirate supernatant completely.
18. For 1 brain (400–500 mg), resuspend cell suspension carefully
with 3100 μL cold PBS.
19. Transfer to a 15 mL tube.
20. Add 900 μL Debris Removal Solution. Mix well.
21. Overlay very gently with 4 mL of cold PBS (pipette very slowly
to ensure that the PBS phase overlays the cell suspension and
phases are not mixed).
22. Centrifuge at 4 C and 3000 g for 10 min with full accelera-
tion and full brake.
23. Three phases are formed. Aspirate the two top phases
completely and discard them.
24. Fill up with cold PBS to a final volume of 15 mL.
25. Gently invert the tube three times. Do not vortex!
26. Centrifuge at 4 C and 1000 g for 10 min with full accelera-
tion and full brake. Aspirate supernatant completely.
27. Optional—remove red blood cells as kit’s instructions.
28. Resuspend in 1 mL cold cutting solution with 1% BSA.
29. Apply the cell suspension to another CellTric 30 μm, placed on
a FACS tube.
Independently of the dissociation protocol, the cells were at the
end resuspended in oxygenated cutting solution with 1% BSA and
filtered through a 30 μm filter into a FACS tube. Dissection of
individual areas was performed according to the Allen Brain Mouse
Reference Brain and Spinal Cord atlas—http://mouse.brain-map.
org/static/atlas.
3.3 FACS Sorting In the juvenile stage, when using mice lines where oligodendrocyte
lineage cells express a reporter gene, FACS sorting the cells, using
stringent gating to avoid collection of myelin debris, produces a
fairly clean cell suspension.
1. FACS sort GFPþ cells using a BD FACSAria III Cell Sorter
B5/R3/V3 system (see Fig. 2 for gate selection).
2. Collect cells in cutting solution with 1% BSA, on ice and
quickly prepare for capture on the C1 Fluidigm system.
3.4 Cell Capture 1. Use cell suspensions in a concentration of 600–1000 cells/μL.

and Imaging 2. Add C1 Suspension Reagent (all “C1” reagents were from
Fluidigm, Inc.) in a ratio of 4 μL to every 7 μL cell suspension.
Fig. 2 FACS gating for exclusion of myelin debris and selection of GFPþ cells from Pdgfra-Cre RCE mice
3. Load 11 microliter of the cell suspension mix on a C1 Single-

Cell AutoPrep IFC microfluidic chip designed for 10- to
17-μm cells, and process the chip on a Fluidigm C1 instrument
using the “mRNA Seq: Cell Load (1772/1773)” script for
30 min at 4 C.
4. Transfer the plate to an automated microscope (Nikon
TE2000E), and acquire a bright-field image (20 magnifica-
tion) for each capture site (Fig. 3) using μManager (http://
micro-manager.org/). The expected time for sorting is approx-
imately 15 min.
5. Manual inspection of each capture site is used to identify empty
chambers, unhealthy cells, or chambers with duplets, triplets,
or more cells (Fig. 3). Only chambers containing a single
healthy-looking cell are further processed. In preliminary
experiments, we had confirmed that cells judged unhealthy
rarely yielded useable cDNA (Fig. 3).
Fig. 3 Example of image analysis algorithm in MATLAB with cells excluded in red
6. After each capture experiment, the individual cell images are

used to determine cell diameter by an automated image analysis
algorithm in MATLAB.
3.5 Library Current single-cell RNA-sequencing protocols vary in the way

Preparation adapter and primers are designed, dictating the information that
will be measured. In our study, we used STRT-sequencing, devel-
oped by the lab of Sten Linnarsson [28–30]. The initial first strand
synthesis is performed with the use of a barcoded oligodT primer,
with a template switching approach. A specific reverse transcriptase
is used, which, when finalizing the first strand synthesis, will add a
sequence of three cytosine nucleotides. A specially designed primer
containing a unique molecular identifier (UMI) sequence is then
added to the mix, to address the intrinsically noisy data of single-
cell sequencing caused by PCR amplification bias. UMIs can then
be used to identify the individual molecules that were present
before the amplification steps. The UMI barcoded primer will
anneal to three cytosines at the 30 end of the first strand cDNA
molecule, with three guanidines. Further elongation will incorpo-
rate the complementary sequence of the template switching oligo-
nucleotide into the existing first strand of cDNA. Subsequently, a
barcode specific for each single cell is added by tagmentation, using

Tn5 transposase, after which library sequencing can be performed.
1. Return the plate to the lab and add 20 μL lysis buffer, reverse
transcription mix, and PCR mix to the designated wells accord-
ing to the manufacturer’s instructions.
2. Place the plate in the Fluidigm C1 instrument and execute the
“mRNA Seq: RT þ Amp (1772/1773)” script. This step
takes ~8.5 h and includes lysis, reverse transcription, and
21 cycle PCR.
3. When the run has finished, harvest the amplified cDNA in a
total of 13 μL C1 Harvesting Reagent and assess the quality of
cDNA on an Agilent BioAnalyzer. The typical yield is 1 ng/μL.
4. Prepare 96 different 10 transposome stocks, each with a
different barcode sequence.
5. Mix six microliters of harvested cDNA with 5 μL tagmentation
buffer, 11.5 μL nuclease-free water, and 2.5 μL 10 transpo-
some stock. Incubate the mix for 5 min at 55 C, then cool
on ice.
6. Wash 100 μL Dynabeads MyOne Streptavidin C1 beads (Invi-
trogen) in 2 BWT, then resuspend in 2 mL 2 BWT.
7. Add 20 μL of beads to each well and incubate at room temper-
ature for 15 min.
8. Pool all fractions, immobilize the beads, and remove the super-
natant (thus removing all internal fragments and retaining only
the 50 - and 30 -most fragments).
9. Resuspend the beads in 100 μL TNT, wash in 100 μL Qiagen
Qiaquick PB, then wash twice in 100 μL TNT.
10. Resuspend the beads in 100 μL restriction mix, designed to
cleave 30 fragments carrying the PvuI recognition site. Incubate
the mix for 1 h at 37 C, then wash three times in TNT.
11. Finally, to elute DNA, resuspend beads in 30 μL ddH2O and
incubate for 10 min at 70 C. Immediately bind beads to
magnet and collect the supernatant.
12. To remove short fragments, use Ampure beads (Beckman
Coulter) at 1.8 volume and elute in 30 μL.
3.6 Illumina High- 1. Quantify the library molar concentration by qPCR using KAPA
Throughput Library Quantification kit.
Sequencing 2. Estimate library fragment length using Bioanalyzer of a ream-
plified (12 cycles) library.
3. Perform sequencing on an Illumina HiSeq 2000 instrument
using C1-P1-PCR-2 as the read 1 primer, and C1-TN5-U as
the index read primer. Multiplex samples, with 96 cells run by
lane. The average read depth per cell is between 200 and
300 thousand reads.
4. Generate reads of 50 bp along with 8 bp index reads
corresponding to the cell-specific barcode. Each read is
expected to start with a 6 bp unique molecular identifier
(UMI), followed by 3–5 guanines, followed by the 50 end of
the transcript.
3.7 Bioinformatic In STRT sequencing, UMIs are located on the 30 end of the cDNA
Analysis molecule. The first sequence read from the original mRNA mole-
cule will therefore be the 50 UTR. This sequence provides sufficient
information to be able to map the transcript to the genome. In
addition, 50 UTR sequencing allows discrimination of specific tran-
scription start sites. Apart from providing unique molecule count
information, UMIs also allow measurement of the amount of PCR
duplication or bias. UMIs make possible to quantify the overall
dynamic range of the library in terms of the rate of new discovered
UMIs per read over time, which should plateau when all the UMIs
have been sequenced in the sample. As such, in the downstream
analysis, we analyze the number of unique molecules (UMIs) rather
than reads. The median amount of UMIs per cell was around 7500
for the whole dataset, mature oligodendrocytes have a median
count of 7500, and oligodendrocyte progenitor cells (OPCs) have
a median count of 4000 UMIs per cell.
3.7.1 Quality Control Many factors within the single-cell RNA-seq pipeline can introduce
bias. Therefore, a quantification of cell quality and bias is para-
mount for successful downstream analysis. Several metrics can be
used when spike-in data (such as ERCC spike-ins, see Subheading
3) is available for the dataset. This includes calculation of the
capture efficiency, calculated by the total spike-in counts captured
within a single sample compared to the total spike counts present.
In STRT sequencing capture efficiencies vary between 10 and 30%
up to 50%, dependent on the cell size and viability [28]. Other
measures include the total counts, total number of genes expressed
per cell, the percentage of mapped counts compared to spike-ins
[31], and principal component analysis (PCA). In most single-cell
datasets, there is a propensity for the first principal component of
PCA to be correlated with number of expressed genes and total
counts per cell. These correlations can sometimes be high (R > 0.9)
and are often unwanted. The backSPIN clustering algorithm (see
below) is less sensitive to these artifacts; however, normalization
schemes might be useful for downstream differential analysis pur-
poses and to quantify technical variation, cell-cycle variation, and
biological variation (see Note 3).
3.7.2 Accounting We mapped individual count reads to genes, resulting in a count

for Technical Variability expression matrix where genes are rows and individual cells are
(Noise) in Single-Cell Data columns. The gene expression matrix is then fit to an estimation
and Feature Selection governed by log2(CV) ¼ log2(ma + k) by calculating the coeffi-
cient of variation (standard deviation divided by the mean) relative
to the mean expression level (Fig. 4). Genes above this line have a
greater chance of demonstrating biological variability [6, 28], and
are therefore used for the first initial clustering with the backSPIN
algorithm, described below.
3.7.3 The BackSPINV2 Many clustering methods for single-cell RNA-Seq data are cur-
Algorithm rently available (see Notes 4 and 5). We used the backSPIN algo-
rithm, which is modified from the sorting algorithm called “sorting
points into neighborhoods” or SPIN [32]. The backSPIN algo-
rithm sorts the distance matrix (often Euclidean distance) into a
one-dimensional ordering for both genes and cells. Splitting of cells
to generate clusters was performed based on the following
parameters:
splitlev ¼ 5; Nfeture1 ¼ 500; Nfeture ¼ 50; N_to_backspin ¼ 150;
N_to_cells ¼ 200; mean_tresh ¼ 0.1; fdr_th ¼ 0.3; min_-
gr_cells ¼ 5; min_gr_genes ¼ 3;
BackSPINV2 uses the splitting algorithm as previously
described [6]. Feature selection based on the coefficient of variance
relative to the mean is performed after each split of the backSPIN
algorithm after which subsequent splits are performed. The
amount of splits is defined by the “splitlev” argument. Manual
inspection of clusters can lead to a merger of subset of clusters, if
Fig. 4 Finding the relationship between gene expression and technical noise. Low mean counts have higher
variation compared to high mean counts, support vector regression fit
inspection of the heatmap does not reveal obvious differences

between the clusters [14] (which means the clusters were over
split).
After this, an estimation of enriched genes for each cell cluster
is performed based on the following criteria:
1 X 1X 1 X
enrichi ,j ¼ Ei , k = Ei ,k ;posfraci ,j ¼ I Ei ,k >0
hk∈j i k∈j N k hk∈j i k∈j
This means that for the cluster j, and each gene i in cell k, the
enrichment of the gene for the cluster is calculated compared to the
whole dataset. Then, the second expression shows the fraction of
cells that express the gene within the cluster.
Then, a ranking of genes is established by
power
S i , j ¼ enrichi, j ∗postfraci , j
Here genes are ranked based on a power value that sets the
weight for the fraction of positive cells in the cluster. We used a
power value of power ¼ 0,0.5,1 was used to rank the genes. A
cutoff value can then be used to rank the x most enriched genes.
3.7.4 Unsupervised Reconstruction of developmental time points is crucial when

Ordering of Cells attempting to uncover differentiation dynamics. Single-cell RNA--
in Pseudotime seq data will provide a snapshot view of the cells contained within
the sample. Often, information regarding the developmental tra-
jectory of cells is lost. This is particularly true when analyzing
samples collected at different time points during development or
data obtained from differentiation studies. Cell ordering algo-
rithms such as the one employed in Monocle [33] aim to recon-
struct the lost differentiation trajectories by ordering the cells along
an artificial differentiation axis called pseudotime. The principle of
Monocle is based on an algorithm established to order microarray
data [34]. Monocle v1.2 reduces dimensionality by means of inde-
pendent component analysis (ICA), next a weighted graph is com-
puted where cells are vertexes and edges represent the distance in
ICA space. Then, a minimal spanning tree (MST) is computed on
the graph, and cells differing from the MST are reassigned to the
MST in a way that minimizes expression differences with neighbor-
ing cells to establish a stable ordering accounting for noise in the
dataset. For full details see [33]. The oligodendrocyte lineage con-
stitutes many intermediate states in the differentiation process as
illustrated by single-cell RNA sequencing in our paper [14]. This
broad differentiation spectrum allows us to reconstruct the putative
differentiation axis with Monocle (Fig. 5).
Fig. 5 Left: minimal spanning tree and pseudotime calculation using Monocle v1.2, right: expression as a
function of pseudotime for several markers of oligodendrocyte development
4 Notes
1. Traditional low-throughput cell capture techniques compatible

with single-cell RNA-seq are micromanipulation and laser-
capture microdissection (LCM) [35]. Micromanipulation
(using for instance a mouth pipette) allows for visual confor-
mation of the cell morphology and quality, which reduces the
likelihood of doublet libraries in downstream procedures.
Micromanipulation has been used for single-cell sequencing
in the context of low cell count experiments, such as analyzing
preimplantation embryo, allowing capture of hundreds of cells
when given enough time. However, low-throughput cell cap-
ture techniques are insufficient and time consuming when
analyzing samples on the tissue level. Therefore, protocols
involving enzymatic dissociation have been developed, an
essential step in achieving high-throughput cell capture.
2. Oligodendrocyte lineage cells represent a quite large popula-
tion of cells in the CNS. As such, they are a relatively easy cell
group to be collected in an unbiased way (without selection).
However, the presence of myelin from P10 onward turns the

analysis by single-cell RNA sequencing of any cell type in the
juvenile/adult mouse brain into a challenging task. For capture
of cells in C1 microfluidic device, a cell suspension as clean and
deprived of debris (cellular or myelin derived) as possible is
required. When analyzing cells other than the OL lineage, a
thorough extraction of myelin debris can be done using Myelin
removal beads (Miltenyi). However, the same approach is
problematic when studying the OL lineage, since the likelihood
of losing mature OLs during the process is high. Therefore, we
found alternatives for this extra step of sample cleaning. One
alternative is density gradients. In Zeisel et al. (2015) [6], when
analyzing cell types diversity in the mouse juvenile cortex and
hippocampus, an albumin-based gradient was used as part of
the Papain Dissociation system from Worthington. Despite the
successful collection of several cells along the oligodendrocyte
lineage with this method, oligodendrocyte precursor cells were
lost in the process of centrifugation, probably due to their small
size (<7 μm) [36] and the low speed used in the gradient
(70 g as opposed to the standard 300 g speed used for
OPC primary culture preparation). Therefore, we further opti-
mized the tissue dissociation protocol to avoid losing both the
OPCs and the mature OLs (methods). For adult or juvenile
CNS tissue derived from non-transgenic mice, an extra gradi-
ent step from the Adult brain dissociation kit (Miltenyi) was
used. This gradient was able, as opposed to the Worthington’s
albumin gradient, to clean the myelin debris without losing
either the OPCs or the mature stages.
3. Estimating sources of variance and normalizing data
Estimating sources of variance in the dataset can be difficult,
even when a principle component correlates with technical
variance or other types of variance, such as the cell cycle. It is
often not enough to simply remove the associated PCA com-
ponent from the dataset. Methods have been developed to
easily infer certain types of variances and even correct for it
[9]. Moreover, normalization has been common practice when
analyzing bulk RNA sequencing. However, normalization of
single-cell data requires different approaches due to the intrin-
sically zero-inflated nature of the gene expression matrix, due
to both technical and biological factors. A normalization
method has been developed that utilizes a deconvolution
approach, meaning normalization of pools of cells by summing
the expression within the pools leading to fewer zeros. Then
pools of cells are “deconvolved” and a size factor estimate is
established for each individual cell [37].
Another random document with
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CHAPTER III
THE FIRST TRANT EPISODE
§1
JUNE sunlight was scorching the tarred asphalt of the Ridgeway,
and Catherine and Helen were sauntering homewards beneath the
heavy trees. Their conversation savoured of “shop.”
“Two hours the last map took me,” said Catherine, indignantly,
“and we’ve got another in less than a fortnight.... Rivers and
mountains as well.... And it isn’t as if North America was easy, either
... there’s all those lakes....”
“I shan’t put in those islands at the top, anyway,” observed Helen.
“I shall leave mine till to-morrow morning,” continued Catherine.
“That is, if I do it at all.... And I shall do it on typewriting paper so I
can trace it.”
“She won’t take it if you do.”
“She’ll have to....”
At the corner of the Post Office the conversation took a personal
turn.
“We’re having a social at the Baptist Church next Saturday. Will
you come?” asked Helen (Helen attended a rather prosperous
Baptist establishment in Upton Rising).
Catherine walked on for some moments before answering. She
seemed to be weighing things up.
“I might,” she answered. Then, as an after-thought, she added: “I
suppose you’ll all be there?”
“Oh yes. There’ll be me and father and mother and Millie,
perhaps the Lester girls as well....”
“George?” Catherine’s voice rather overdid itself in the effort to
appear casual. Helen looked at her keenly.
“Possibly,” she replied, in a voice that might have meant
anything. There came a rather curious pause. They had reached the
corner of the High Street before Helen spoke again.
“So that’s it, is it?” she remarked, as they crossed the tramway
junction.
“That’s what?” said Catherine gruffly.
“That’s what’s been making you so ... so different—lately.... I’d
been wondering what it was. I never guessed it was George.”
“How did you find out?”
“I didn’t find out. You just told me.”
Catherine turned down Hanson Street, the road immediately
opposite the Ridgeway.
“Let’s go down here,” she suggested. “It’s quieter. I can see
you’ve a lot to say to me.”
Helen took her arm.
“No, I haven’t.... I don’t know that I can say anything, really....
Only I think you’re silly.”
“Why?” The word rang out like a pistol-shot.
The reply did not come immediately. When it did it sounded limp
and uncertain.
“Because ... because you’ll be disappointed in him.”
“What’s the matter with him, then?”
“Nothing much. He’s all right ... only ... he’ll disappoint you, one
way or another. He’s not as clever as he seems. Besides——”
“Yes?”
“He doesn’t like you.”
“He doesn’t? Has he told you so?”
“Not in so many words. But I know. He may like you to flirt with,
but he doesn’t like you. My advice is, if you’re getting serious, give
up the flirting. With him, at any rate.... After all, you can always find
plenty of chaps to flirt about with....”
(Her father had said “fellers.” She said “chaps”!)
“But I don’t want them, maybe.”
“Well, go without them, then.” (They were at the corner of
Kitchener Road.) ... “I never thought much of flirting about as a
pastime.”
It was a curiously elliptical conversation throughout, and at the
gate of No. 24 they both seemed eager not to prolong it by standing.
They said good-bye immediately, and both were conscious of
electricity in the atmosphere.
That evening Catherine found herself unable to concentrate on
homework. Mr. Weston was out at night-school, and she was thus
left alone in the house. The nine o’clock rule was now virtually
inoperative, since her father did not return till half-past ten on three
nights out of the week. At about ten past nine Catherine put aside
her books and went out for a walk. She had finished all her work
excepting the map.
Cubitt Lane at this time on a glorious June evening was full of
courting couples. They lurched along in a peculiarly graceless
fashion, each leaning against the other.
“I wouldn’t do that,” thought Catherine, virtuously. “That is silly, if
you like.”
At the bridge over the railway she heard a brisk “good evening”
addressed to herself. She turned and saw it was George Trant....
“Where’re you off to?” he asked good-humouredly.
“Taking a walk.”
“So’m I.... Let’s go up the road....”
“All right.” ... They climbed the hill past the King’s Arms, and
entered the Forest.
§2
The first leaves of autumn were beginning to fall when Catherine
returned to Bockley after a fortnight at Hastings. Day after day of
glorious September weather had covered her cheeks and arms and
hands with freckles: her hair, too, was fluffed and shining with
continual sea-bathing: her general appearance was rather wild and
undomesticated for such a place as Bockley. She returned on
Saturday night, and Sunday found her waiting outside the Baptist
Church at Upton Rising. Evening service was over at eight o’clock,
and she judged that Helen would be there.
Helen greeted her at the church door.
“Only you?” said Catherine.
Helen nodded. “The others went for a walk.... It’s a fine night—
let’s take a tram to the Forest.”
The trams of the London County Council ran along the end of the
road. They boarded one; it was full, and they had to stand on the top.
“You look well,” remarked Helen.
“Oh, I’m all right,” replied Catherine, and the conversation
languished.
What ensued after that would always in Catherine’s mind be
inextricably bound up with the sway and purr of trams along the high
road.
“George has gone away,” remarked Helen, à propos of nothing.
“Oh?”
“His firm’s given him a job in Manchester. A good opening, it
seems.... I got a letter from him yesterday. He enclosed a note for
you: I suppose he didn’t know your address.... I believe I’ve got it on
me....”
She fished in her hand-bag and extracted an envelope, from
which she took a folded half-sheet of paper and handed the latter to
Catherine.
It was rapidly getting dusk, but the lights in the tram were not yet
lit. On every alternate tramway standard hung an arc lamp, and
these were now fizzing and spluttering into pale brilliance. Catherine
read the note (it was roughly written in copying pencil) in quick
spasms as the car swirled along.
my dear cathie,
As you will perceive, I have got shifted to Manchester, where I
shall no longer have the pleasure of your delightful society, which, as
you will not doubt, is a great loss to me personally. However, I am
likely to enjoy my stay here: there are some splendid girls working in
the same office with me, though none of them has your own
Inimitable red hair. If there is one thing I regret it is that the before-
mentioned red hair has occasionally led me to say things I did not
mean and to do things I did not mean to do. I am sure that you, with
your wonderful capacity for understanding, will grasp what I am
trying to sketch out. We have had some interesting discussions
together during the last few months, and for these at least (not to
mention the spiritual inspiration given me by the passionate flame of
your hair) I am deeply grateful.
I hope you will always believe me to be what I am, viz., your
sincere admirer,
george trant.
P.S.—My lodgings are not permanent, so there would be little
point in enclosing my address.
Catherine was slow to grasp the full meaning of the note. As it
dawned upon her her lips tightened, and she gripped fiercely the rail
against which she was leaning. The tram lurched to a standstill, and
there was the usual scramble to get down the stairs. “High Wood,”
the conductor called out.
“Come on,” said Helen, and they descended.
In the Forest glades the night air was cool and sweet. For some
distance they walked on in silence. Catherine was the first to speak.
They had reached a clearing, and under the open sky the daylight
still lingered.
“I daresay you’d like to read it,” said Catherine. She held out the
note at arm’s length.
Helen gave a queer ejaculatory laugh.
“I’ve already done so,” she said.
“What?”
“Oh, I know it’s not quite the thing to read other people’s letters....
But I wanted to know what ... what he would say to you, and I
thought perhaps you wouldn’t show me.”
Catherine crumpled up the note and put it in her pocket.
“Well, you know, anyway,” she said gloomily.
They passed again into the cool Forest glades.
“I was right,” said Helen, quietly. “I knew he’d write you something
like that. He’s good at that kind of letter-writing ... sort of cheap
cleverness he excels at I’d half a mind not to let you see it.”
There came a long pause. They had reached the high road to
Chingford before it was broken.
Catherine suddenly took the crumpled letter from her pocket, and
began tearing it up into minute fragments.
“See,” she cried passionately, “you can tell him this is what I did
with his letter I You can tell him there’s better fellows in the world
than he is, and Cathie Weston isn’t going to break her heart over
him! ... Tell him I’m not a soppy little schoolgirl.”
She flung the pieces on the ground, and began stamping on
them.
“You’re being silly,” said Helen, quietly.
“And tell him,” went on Catherine, “that if he thinks he’s under an
obligation to me, he’s made a mistake. I’m grateful to him—for letting
me see what he really is.”
Her words rattled like the passage of a lorry over granite setts.
“Come on,” said Helen, “we’ll get to Chingford, and take the train
back.”
“You’ll tell him?”
“I don’t promise. I think you’d better forget all about him ... after
all, you can’t do anything....”
“I don’t want to! I merely want him to know that I don’t mind.”
“Well, that’s all right, then. He’ll know that if he hears nothing from
you.”
“He won’t. He’ll think he’s left a broken-hearted girl to cry over
him.”
“I don’t think he will.”
“... because I don’t believe in being broken-hearted. I don’t think
it’s possible to die of a broken heart. I’m certain I shan’t, anyway. I
won’t let any man mess about with my life. It’ll take a pretty big
misfortune to make life not worth living to me. If he’s tired of me I’m
just as tired of him. Tell him that!”
“This way ...” said Helen, guiding her into the Station Road. “We’ll
just be able to catch the 9.45....”
§3
Helen left the train at Upton Rising, but Catherine went on to
Bockley. The Town Hall struck the hour of ten as she was walking up
the station approach. At this time the crowds along the High Street
were beginning to disperse; the trams and buses were full of
returning excursionists. Neglectful of the time and with no very
definite aim in view, Catherine turned into the Ridgeway. It was
directly opposite to the quickest way home, but its shady avenues
and flower-scented front gardens suited her mood better than the
stark frowsiness of Hanson Street. Her mind was in flux. She did not
know whether what had happened was going to be an important
stage in her life or not. She did not know how much of her feeling
was disappointment, and how much was mere wounded dignity. She
could not estimate the depth of the feeling she had had for George
Trant. It seemed inconceivable that she had ever been in love with
him....
She started to administer to herself wholesome correctives. “It’s
no good,” she told herself brutally, “your imagining yourself the
heroine of a tragedy, suffering more poignantly than ninety-nine
people out of every hundred, because it’s not the truth. What you are
feeling now is felt sometime or other by the majority of all people:
there’s nothing a bit singular or exceptional in your case. It’s a
mistake to pride yourself on suffering more exquisitely than other
people.”
Then she poured cold logic over herself.
“He’s only one man among millions, and in no sense is he
markedly superior to the average. A certain spurious cleverness, a
talent for mockery, a deft finesse in expressing cruel things in soft
words ... absurd that he should become so much to you or to any girl
... there’s nothing admirable in him, therefore you are lucky to get rid
of him.”
It sounded convincing enough.
She walked on, scarce heeding whither she was going, and all
the time her mood alternated between stormy resentment and cold
self-reproach. There were moments too of grey hopelessness, and it
was only her constantly recurring indignation that swept her out of
these. Every inch of the roads she traversed was associated with
him: every gate and tree seemed to call out in mocking melancholy
—“This was where ... this was where....” Not a street corner but was
inextricably bound up in her mind with some remark of his and the
exact phase of their relationship when he had uttered it....
Under heavy trees that split the moonlight into a thousand
fragments she suddenly heard the rich hum of a grand piano. She
stopped. She stood in the shadow of the hedge and listened in
rapture. The house was a large one, with a corner bay-window wide
open, and it was from that room evidently that the music was
proceeding. It was some rapid piece full of rippling streams of notes
with very few chords, octaves in the base clef that thundered like the
oncoming tide, swirling waves of treble triplets that were light as air,
yet beneath all the laughter and freedom, a sense of dim, unuttered
passion, half hopeful, half melancholy. Long afterwards she knew it
was Chopin’s Black Note Study in G flat. But then it had no name to
her. It might have been the latest ragtime craze for all she knew: all
she cared was that it expressed all the feelings in her own heart that
she had thought inexpressible, things that she had often and in vain
tried to wring out of the Collard and Collard at home. At that moment
it is probable that she would have given everything she had in the
world for that piano. It stood to her as the one way to salvation. She
would have bartered her soul for it. As it was, she stood there in the
spattered moonlight and cried for it. At any rate, she cried.... The
piece finished up in a tremendous cascade of double octaves, and
she waited nearly half an hour after that, hoping the playing might
begin again. Then she walked back to Kitchener Road almost in a
state of trance. The Bockley High Street was very white and
deserted, and far into the dim distance stretched the tram-rails, blue
and infinite. It was long past eleven. But Catherine was dreaming—
dreaming of one thing only (though that one thing was strangely
complicated by other things)—dreaming of a grand piano, dreaming
of the ecstasy of playing it as she had heard it played that night. The
vision of her ambition came to her as she turned into Kitchener
Road. She would become a great pianoforte player. Already
discerning critics—adjudicators at musical festivals and such like—
had prophesied a career for her if she would work hard. Hitherto it
had not seemed worth while to work hard. Now it became suddenly
and tremendously worth all the soul and energy she could give to it.
Nothing else mattered. Nothing else could ever matter. Whatever
stuff her soul was made of, music was part of it, and music would
answer everything her soul asked.
At home her father was waiting up, vaguely remonstrative as
usual.
“Worse and worse it gets, Cathie ...” he began ... “the first night
you’re home after your holiday you land in at twenty to twelve! ... it’s
not good enough ... you’ve had all the morning and afternoon. I can’t
think what makes you want to go walking the streets this time....”
“I’m not having any supper,” she said brusquely. “Good-night....”
“But——”
“Oh, don’t worry ... I’ve had some,” she lied. As she fled upstairs
she heard him murmuring something. A great change had come over
him since his wife died. He had been getting ever slower and feebler.
It was becoming more and more evident that it had been only his
wife’s incessant nagging that had spurred him to the minimum of
activity. Now he pottered aimlessly about the garden. His
attendances at the Duke Street Chapel became more and more
infrequent, and finally ceased altogether. People said (often
facetiously) that he was pining away of grief at his wife’s death. It is
doubtful if this were a complete diagnosis....
Up in the little back bedroom Catherine did a thing which she had
not done for a long time. She prayed. Ch-artinevin was no longer a
choleric old gentleman with white side-whiskers and a devouring
passion for adulatory worship. He had long ago ceased to be that,
and he had not begun to be anything else. Catherine, though she
never altogether recognized her position, had no very definite belief
in either Him or the rest of the accepted doctrines of Christianity. She
prayed, not out of religious fervour, but from a variety of complex
motives, one of which was certainly a desire to straighten out her
own ideas by reducing them to more or less coherent form. Among
other things, she prayed for a grand piano. “Lord, give me a grand
piano,” was her unorthodox variant upon the more usual bedtime
supplications. “Lord, do give me a grand piano,” she pleaded. It is
curious, but she did not in the least expect the Lord to take any
notice. She was even doubtful whether the Lord were listening. Yet
she kept on repeating the demand for a grand piano. Also she
decided how she would catalogue the whole George Trant episode.
It was nothing. It was to be regarded as nothing. Tears broke in upon
her decision to regard it as nothing. The grand piano and all that it
meant to her kept looming on the horizon. Then she felt a little
ashamed of crying. “I never used to cry,” she thought. “Not even after
a sound thrashing.” She tried to calm herself. “I’m getting soppy,” she
reflected. “Crying like a little kid. All because of that piano. That’s
what done it....” It was long past midnight when she fell into troubled
sleep.
CHAPTER IV
NOCTURNE
§1
ON a certain bitterly cold night in November, Catherine stood on the
doorstep of No. 24, Kitchener Road, with her overcoat and hat in her
hands. Despite the chilliness of the atmosphere her cheeks were hot
and flushed, and her sensations took no notice of the blustering wind
that raged along the road. For several moments she stood still on the
doorstep, with heaving breast and head flung back defiantly. Then,
still carrying her hat and overcoat, she went out into the street,
omitted to shut the gate behind her, and walked at a terrific pace in
the direction of the Bockley High Street.
It was eleven p.m. Her steps rang loudly along the deserted
pavements; occasionally she lurched forward as if desiring to
increase her pace, and this disturbed the rhythmic beat of her steps.
She passed nobody, except at the junction of Hanson Street, where
a couple of belated revellers slunk past with the furtive attitude of
those who know they ought to have been home long since. They
were too intent upon their destination to notice her. Only where there
were large front gardens did her passing excite attention, and here
congregations of cats, gathered for midnight revelry, dispersed with
mournful sound as her footsteps approached.
At the corner of the High Street she stopped. It seemed to occur
to her for the first time that to carry one’s hat and overcoat upon
such a night was in some degree unusual. With careful deliberation
she put them on. Then she laughed softly, and her laugh was a
strange mingling of rapture and defiance. That which she had
thought impossible had come to pass. After years of undeviating
placidity fate had at last done something dramatic with her.
She had been turned out of the house at No. 24, Kitchener Road.
Her father had done what he had never before been known to do:
he had lost his temper, and lost it thoroughly.
He had said: “My God, Cathie, I won’t stand that! ... Out you go!”
He had pushed her into the lobby, and while she was reaching for
her hat and coat he had struck her on the face with the back of his
hand.
“Out you go!” he repeated, and Catherine saw that his temper
had not yet reached its height. “I’m done with you! ... Are you going?”
He actually picked up an umbrella and began brandishing it with his
hand grasping the ferrule.
Catherine had opened the front door in vague terror of what he
was going to do. The door was banged after her with a vicious kick
from within. Then her cheek where he had struck her began to
hurt....
§2
The cause of the altercation had been Catherine’s determination
to accept a situation which he did not wish her to accept. She had
answered the advertisement, interviewed her prospective employer,
and received word that she had been appointed before even
mentioning the matter to him. Then at teatime on a Friday afternoon
she casually remarked:
“By the way, I’ve decided to get some work.”
He looked up at her as if the word were unfamiliar to him.
“Work?” he said, astounded. “What do you mean?”
“I mean I’ve applied for a job and been offered it.”
He seemed to have difficulty in comprehending what she said.
“A job? What job?”
“They want a pianist at a cinema. Good salary. Only work in the
evenings....”
“But, my dear girl——”
“Well?”
“Don’t cut me short like that.... I was about to say....”
“Oh, I know what you’re about to say. You’re hopelessly against
it, aren’t you?”
“Well, if I am, you——”
“Why are you?”
“I do wish you’d give me time to speak, Catherine. You spring this
on me so suddenly.... I had no idea you were ever thinking of such a
thing, to begin with. Even now it seems incredible to me. I can’t
understand it.”
“Can’t understand what?”
“Why you want to do it ... it’s ... it’s unnecessary. Haven’t you
enough money?”
“Oh, it’s not a question of money. I want to have some work to do,
something to get interested in.”
“But you have the work of the house to carry on with. Surely
that’s enough.”
“Oh, that’s enough. In fact, that’s a great deal too much. I’m sick
and tired of housework. Some girls may like it, but I don’t. I’d sooner
pay some girl who likes it to do it for me. Besides, I want to be
independent.”
He gave a start of surprise. “What’s that you said?” he asked,
incredulously.
“I said, independent.”
There was a tense pause.
“Somebody’s been putting some silly modern ideas into your
head. All that bosh about independence, I mean. A girl’s place is in
the home, when she’s got one. Until you make a home of your own
your place is here.”
“I suppose you think I ought to get married.”
“Married? ... Heavens, no! ... You’re only nineteen! Why, I never
even met your mother until I was twenty-four! Don’t you worry your
head about marriage. Let it alone until the right feller comes along. I
expect you’ve been reading too many trashy novels lately, that’s
what it is.”
An angry light leapt into her eyes.
“Well, if you think I’m going to scrub floors and wash dishes until
the right feller comes along, as you call it, you’re jolly well mistaken. I
wouldn’t do it even if I was sure the right feller would come along. I’m
not made that way. I want a bit of liberty. I want to live.”
“My dear Catherine, you have everything you need. I can’t see
what you’re making all this fuss about. Really I can’t.... You’re a good
deal better off than some girls, I can tell you. What about poor Nellie
Selborne and——”
“Oh, what on earth have they got to do with it?”
“Well, if you won’t listen to me, I suppose ...” He waved his hand
deprecatingly. “Suppose we stop arguing. Let’s hold the matter over.
I’m certain that with a few days’ thought you’ll——”
“But I can’t hold the matter over.”
“Why not?”
“Because the situation’s been offered me. I’ve either got to
accept it or reject it on the spot.”
“Well, Catherine, I’m sorry to go against you, but it will have to be
so, in this case. Understand, I mean it. I mean to have my own way
in this matter. I won’t have you strumming away every night in a
third-rate picture house. I’m going to put my foot down firmly in this
matter. You must reject the offer.”
He made a gallant but not entirely successful attempt to appear
dignified by resuming the perusal of his newspaper. Catherine bit her
lip and went a little pale.
“That’s a pity,” she said quietly.
“Why is it a pity?”
“Because I’ve decided to accept it.” Her lips were tight, and there
was the suggestion of restrained emotion in her voice.
Something happened to his eyes. They opened terrifically wide
and gazed at her expressionlessly for several seconds.
“What’s that?” he said.
His eyes unnerved her somewhat. But she steeled herself to
repeat her ultimatum.
“Because—I’ve—decided to—to accept.”
Pause. “That’s all,” she added, irrelevantly, as if by way of
clinching the matter.
Another pause. The clock tactfully struck in with the
announcement of six o’clock. That seemed to break the spell. He
rose and made for his hat.
“H’m,” he ejaculated, sharply. “I see. That’s what it amounts to, is
it.... Well, you’ll have time to think it over. I’m off to school now.”
He took a sheaf of night-school exercises from his desk and
stuffed them in his pocket. Not another word came from him.
Catherine was almost hypnotized by his quick, startling movements,
so unlike his usual apathy. He strode firmly down the lobby and shut
the door after him more noisily than usual. She could hear his
footsteps along the street, and he was walking at a pace that was for
him unprecedentedly rapid. When he was quite out of hearing she
sank down into the chair he had just vacated. The tension of the
argument had given her a sense of physical exhaustion. Yet
spiritually she was thrilled by a strange feeling of exhilaration: it
seemed to her that after an interval of drudgery she was once again
being drawn into the vortex of momentous happenings. She was
absolutely certain of one thing: she would not give way. If he chose
to make her disobedience a “test-case” of the father’s right to inflict
his will upon the daughter she would await whatever steps he took
with calmness and determination. But she would never give way.
She was nineteen, and to her nineteen seemed old age. Things he
had said in the course of the argument had annoyed her
inexpressibly. They were little things, mostly. Bringing in the case of
Nellie Selborne, for instance, was silly and entirely irrelevant. Nellie
had paralysis down one side, and existed apparently for the purpose
of proving to all other girls how lucky they were. Then again,
Catherine disliked intensely his massive declaration that “a girl’s
place is in the home.” He had talked about “waiting for the right feller
to come along,” and this passive method of getting through life
roused all the scorn and contempt in her nature. Also he had talked
about her “strumming in a third-rate picture house.” It was typical of
him to assume that it was third-rate before he had heard even the
name of it. He had been ridiculously unfair....
She went over to the writing-desk where he marked his school
exercise books. Something within her said: You are angry and
excited now, but you will soon cool down and then probably you will
give in to him.... To this she replied passionately: I won’t give in to
him.... But, continued the part of her which always told the truth, you
will give in to him if you wait till your temper has cooled down....
Better write now accepting the situation, and post it before he comes
back from night-school. Then the matter will be really settled. Then
you can say to him when he comes in: “It’s no use arguing about it
any more. I’ve written to accept the job. The thing’s done now and
can’t be undone.”
She wrote the letter as quickly as she could, for the feeling of
supreme depression, the feeling that she was doing something
regrettable and irretrievably silly, was becoming heavier upon her
every second. She was just addressing the envelope after fastening
it when she heard the key fumbling at the front door. For the moment
a kind of panic fear seized her. He was coming back. He must have
turned back before reaching the school. His footsteps down the
lobby sounded brutal and unnecessarily noisy. She swung round in
her chair and sat awaiting his entrance with the penholder stuck
between her teeth. The half-addressed envelope lay on the desk
invisible behind her back.... He flung down his hat and coat on the
table.
The moment was so tense that Catherine spoke merely to
interrupt the horrible silence of it.
“Was there no school to-night?” she asked, with an effort to
appear perfectly casual.
“I’m not going,” he snapped curtly, and took down the red-ink
bottle from the corner of the mantelpiece. That meant he was going
to spend the evening marking exercise books.
She was thoroughly frightened. Her mother’s tempers and tirades
had never frightened her, because she was used to them and knew
them intimately, as a doctor knows the illness of a familiar patient.
But her father was normally so quiet and placid and mild-mannered:
she had never seen him in a temper, although when she was a little
girl, boys who were in his class at school had told her that on rare
occasions he got “ratty.” But she had never known him in such a
condition. In this phase he was a complete stranger to her. And she
was apprehensive, as she would have been if a stranger had
entered the house when she was alone.
He came to the desk to get his exercise books. She thought at
first he was going to strike her. But he merely leaned over her and
lifted the lid. As he did so he must have seen the half-addressed
envelope lying on the top. But he did not say a word. His silence was
unnerving.
Always he used the desk for marking exercise books. But this
time he arranged the pile of books and the pen and ink on the dining-
table.
“You can use the desk,” he said curiously, “if you’re wanting to.”
His politeness, his unusual solicitude for her comfort, was horrible!
Normally, if she had been at his desk, he would have said: “Now look
here, Cathie, it’s too bad of you to want to use my desk when I want
it. After all, it’s my desk. You’ve got all the day to use it when I’m out.
Can’t you use the table?”
She would have understood a speech like that. But for him to say
so thoughtfully, so obsequiously, “You can use the desk if you’re
wanting to,” was charged with all the nameless horror of the
unprecedented.
It was half-past six. The clock struck. He was assiduously and
seemingly quite normally putting red-ink ticks and crosses on
algebra sums. Yet she knew that the atmosphere was very far from
being normal. She took a book from the shelf and sat down in the
chair by the fire, but it was difficult to read. She could hear the ticking
of the clock and the steady scratching of his pen, and flipping of
pages. He went on for hours. When he had finished one pile of
books he went to his desk and fetched out another. Then again, if he
had not done so the first time, he must have seen the envelope with
its incomplete address. But he went on with his work at the table.
Supper time came, but he made no sign of clearing away his books.
And then his surliness and sulkiness, whichever it was, ceased to
frighten her, but began to annoy her acutely.... The last post went at
eleven-thirty. Come what might she would post that letter. At five
minutes past eleven she went over to the desk with the intention of
finishing the address. She had got as far as the “p” in “Upton” when
she saw that he was regarding her intently. As soon as he saw that
she had noticed his glance he put down his pen and swung back on
his chair.
“Now then, Cathie,” he began brusquely, “this matter’s got to be
settled.... You understand. No nonsense. What’re you going to do?”
She bit the end of the penholder.
“I’m going to accept the thing,” she said firmly, though she had
difficulty in restraining her apprehension and excitement.
“You’re not!” he cried, advancing menacingly. “Understand, I
forbid it! I’m going to be firm in this business. You’re not to accept
that situation. D’you hear?”
He picked up the envelope she had been engaged upon. She
knew that he had seen it before. But he pretended not to have done.
She despised him for that little perfidy.
“What’s this?” he cried, snatching it up vehemently. Then he
pretended to realize. “You’ve been writing to accept it?”
“Yes.”
For a moment she thought he was going to do her physical
violence. Then he tore the envelope across and flung the two pieces
into the fire.
“Oh, that doesn’t matter,” she said contemptuously, “that’s merely
childish. I can easily write another.” (In her anger she did not
remember an occasion when she had been smitten with the same
kind of childishness).
It was then that he cried: “My God, Cathie, I won’t stand that! ...
Out you go!”
§3
At the corner of the Bockley High Street her only feeling was one
of nervous jubilation. The clock chimed the quarter. She
remembered with a little thrill of ecstasy how on all other occasions
at night when she had heard the clock chime a quarter past eleven
she had been anxiously wondering what sort of a row there would be
when she reached home. Now she was free. She was not returning
home. She was leaving. She was free to go where she liked and do
what she liked....
If it were summer time, she thought, I would walk to the Forest
and sleep out under the stars....
But it was November.... She decided to travel up to the City and
spend the night in one of the waiting-rooms at the big terminals. The
next day she would look out for lodgings.... Money was a difficulty. In
her pocket was a purse containing the residue of the week’s house-
keeping money. It amounted to five and sevenpence half-penny.
There were also a couple of penny stamps....
The ideal time for this enterprise would have been a Monday
evening in June or July.
Still, she would have to make the best of it. With light step she
passed along the wide expanse of the High Street in the direction of

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Oligodendrocytes: Methods and

Protocols David Lyons

Plant Bioinformatics Methods and Protocols 2nd Edition

Modern Methods of Construction and Innovative Materials

Zebrafish Methods and Protocols Koichi Kawakami

SNAREs: Methods and Protocols Rutilio Fratti

Phytoplasmas: Methods and Protocols Rita Musetti

Metalloproteins: Methods and Protocols Yilin Hu

Nanotoxicity: Methods and Protocols Qunwei Zhang

Autoantibodies: Methods and Protocols Gunnar Houen

For further volumes:

Methods and Protocols

David A. Lyons and Linde Kegel

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Edinburgh, UK David A. Lyons

13 A Drug-Inducible Transgenic Zebrafish Model for Myelinating

SYLVIA AGATHOU  Department of Veterinary Medicine, Wellcome Trust-Medical Research

CHAITALI MUKHERJEE  Institute of Neuronal Cell Biology, Technical University Munich,

Single-Cell RNA Sequencing of Oligodendrocyte Lineage

The advent in the last decade of transcriptomic technologies, cou-

investigated heterogeneity within the oligodendrocyte lineage in

2.2 Tissue 1. Pasteur glass pipettes.

2.3 FACS Sorting 1. FACS tubes—Corning, catalogue number #352058.

2.5 Lysis, Reverse 1. Oligonucleotide sequences:

2. Lysis buffer: 0.15% triton X-100, 1 U/μL Takara Rnase inhib-

2.6 Bioinformatic 1. MATLAB software—MathWorks.

2.7 Tagmentation 1. 96 different 10 transposome stocks (6.25 μM barcoded adap-

4. BWT: 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 2 M NaCl,

2.8 Illumina High- 1. KAPA library quantification kit—KAPA biosystems, catalogue

Single-cell RNA-Seq has been shown to be a powerful tool to

3.1 Tissue Collection Tissues were collected in oxygenated 1 cutting solution.

dissociation, such as FACS sorting with strict gating parameters to

18. Centrifuge cell suspension at 300 g for 10 min at room

15. Apply the cell suspension to a MACS SmartStrainer 70 μm,

3.4 Cell Capture 1. Use cell suspensions in a concentration of 600–1000 cells/μL.

3. Load 11 microliter of the cell suspension mix on a C1 Single-

6. After each capture experiment, the individual cell images are

3.5 Library Current single-cell RNA-sequencing protocols vary in the way

barcode specific for each single cell is added by tagmentation, using

3.7.2 Accounting We mapped individual count reads to genes, resulting in a count

inspection of the heatmap does not reveal obvious differences

3.7.4 Unsupervised Reconstruction of developmental time points is crucial when

1. Traditional low-throughput cell capture techniques compatible

However, the presence of myelin from P10 onward turns the

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SYLVIA AGATHOU Department of Veterinary Medicine, Wellcome Trust-Medical Research

CHAITALI MUKHERJEE Institute of Neuronal Cell Biology, Technical University Munich,