GSM7897840: 10 clones, pooled scRNA-seq; Homo sapiens; RNA-Seq
One day before differentiation, each of 10 hESC barcoded clones were counted, mixed at the same cell number ratio and then seeded at a density of 2.3 × 105 cells/cm2 onto 12-well cell culture plate. At DE and PP2 stages, pooled differentiating cells were dissociated into single cell suspension by TrypLE Select for 5 min at 37 °C. Cells were then stored in BAMBANKER™ freezing medium for future experiments. For scRNA-seq, frozen cells were thawed and sorted to collect live GFP+ cells. Cellular suspensions were then loaded on a Chromium Controller following the manufacturer's instructions (10x Genomics Chromium Single Cell 3′ Reagent Kit v3.1 User Guide). cDNA libraries and targeted LARRY barcode libraries were generated separately using 10ul cDNA each. cDNA libraries were made under manufacturer's instructions and targeted LARRY barcode libraries were amplified using specific primers (F: CTACACGACGCTCTTCCGATCT; R: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtaaccgttgctaggagagaccataT). Individual LARRY barcode constructs were cloned from LARRY barcode library (Addgene:140024) and transfected to 293T cells to generate lentivirus. Next, each KO and WT hESC clone was infected with a unique LARRY barcode at low MOI. One week after lentiviral infection, the barcoded cells, which expressed GFP, were sorted out and cultured in E8 medium as described in previous section.