Replace medium after 12 hours (Day 2). #####%%%%% output "Digest mESCs" are used in line4
{"action": "replace", "output": "Digest mESCs", "container": ["medium"], "volume": [""]}
Digest mESCs with 0.05% trypsin, prepare for FACS into 96-well plates (Day 10). #####%%%%% output "sgRNAs analysis (SPH primers)" are used in line8
{"action": "digest", "output": "mESCs", "reagent": ["0.05% trypsin"], "container": ["96-well plates"]}
Remove single colonies from 96-well plates to 24-well plates, .
{"action": "remove", "output": "", "container": ["24-well plates"]}
confirm positive colonies by transient transfection of sgRNAs analysis (SPH primers) (Day 14-15) #####%%%%% output "positive colonies" are used in line18
{"action": "confirm", "output": "positive colonies", "reagent": ["sgRNAs analysis (SPH primers)"]}
Replace medium after 12 hours (Day 2).
{"action": "replace", "output": "", "container": ["medium"], "volume": [""]}
Sort single cells into 96-well plates by FACS, .
{"action": "sort", "output": "single cells", "device": ["FACS"], "container": ["96-well plates"]}
confirm insertion by PCR (Day 18)
{"action": "confirm", "output": "", "reagent": [""]}
Remove single colonies from 96-well plates to 24-well plates, .
{"action": "remove", "output": "", "container": ["24-well plates"]}
confirm positive colonies by PCR (Day 22) #####%%%%% output "positive colonies" are used in line18
{"action": "confirm", "output": "positive colonies", "reagent": ["positive colonies"]}
Measure fluorescent intensity of colonies by FACS, take fluorescence images under confocal microscope (Day 27). #####%%%%% output "colonies" are not used
{"action": "take", "output": "fluorescence images", "device": ["confocal microscope"], "container": ["colonies"]}