20-30% cell confluent at day 0

Puromycin 0 1 2 3 4 5
(ug/ml)

Day 0

Day 1

Cell did not all die at all puromycin concentration

Day 2

Cell all die at 4 ug/ml puromycin

Day 3

Cell all die at 2 ug/ml puromycin

Day 4

Cell all die at 1 ug/ml puromycin

50% cell confluent at day 0
Puromycin 0 1 2 3 4 5
(ug/ml)

Day 0

Day 1

Cell did not all die at all puromycin concentration

Day 2

Cell did not all die at all puromycin concentration

Day 3

Cell all die at 2 ug/ml puromycin

Day 4

Cell all die at 1 ug/ml puromycin

80% cell confluent at day 0
Puromycin 0 1 2 3 4 5
(ug/ml)

Day 0

Day 1

Cell did not all die at all puromycin concentration

Day 2

Cell did not all die at all puromycin concentration

Day 3

Cell all die at 3 ug/ml puromycin

Day 4

Cell all die at 2 ug/ml puromycin

Day 3 after puromycin

20-30% cell confluent at day 0

Puromycin 0 1 2 3 4 5
(ug/ml)

Cell all die at 2 ug/ml puromycin

50% cell confluent at day 0

Cell all die at 2 ug/ml puromycin

80% cell confluent at day 0

Cell all die at 3 ug/ml puromycin

Day 4 after puromycin

20-30% cell confluent at day 0

Puromycin 0 1 2 3 4 5
(ug/ml)

Cell all die at 1 ug/ml puromycin

50% cell confluent at day 0

Cell all die at 1 ug/ml puromycin

80% cell confluent at day 0

Cell all die at 2 ug/ml puromycin

- After 3 days:
+ 20-30% cell confluent at day 0 group: Cell all die at 2 ug/ml puromycin
+ 50% cell confluent at day 0 group: Cell all die at 2 ug/ml puromycin
+ 80% cell confluent at day 0 group: Cell all die at 3 ug/ml puromycin but almost cell die at 2 ug/ml puromycin
- After 4 days:
+ 20-30% cell confluent at day 0 group: Cell all die at 1 ug/ml puromycin
+ 50% cell confluent at day 0 group: Cell all die at 1 ug/ml puromycin
+ 80% cell confluent at day 0 group: Cell all die at 2 ug/ml puromycin

-> Choose 1; 1.5; 2 ug/ml puromycin for 3-4 days

 Primary DP cell Electroporation

12/21 12/22

Transfection Puromycin
- Centrifuge cell -> cell pellet -> put PBS count cell -> separate into 4 tube -> centrifuge -> cell pellet -> 100 uL R buffer each tube

- Prepare machine: put cuvette in, prepare tip, pipet, 3ml E2 buffer into cuvette

- Prepare PBS in 4 E tube to wash the tip

- Put 900 ul Promo media in 12 well plate

300 uL each tube

GRNA (ug) 12
750
Cas9 (ug) 24
750 + 6 ug GFP
R buffer (ul) 1500

- Pick 100 ul cell DNA mix (no bubble)

- Insert pipet to the cuvette
- Electro
- Release cell into prepared media in 12 well plate
- Wash 5 times in PBS
Cell number: 105 cell/well

Green fluorescent protein

Normal light M Cherry fluorescent protein Merge
(GFP)

Control

Day 1

GFP
Cell number: 105 cell/well
Green fluorescent protein
Normal light M Cherry fluorescent protein Merge
(GFP)

Control

Puromycin 1 ug/ml

GFP

Control
Day 3

Puromycin 1.5 ug/ml

GFP

Control

Puromycin 2 ug/ml

GFP
Cell number: 105 cell/well

12/21 12/22 12/24

Transfection Puromycin Puromycin

Green fluorescent protein M Cherry

Normal light Normal light Merge
(GFP) fluorescent protein