GSM7230012: hCiPSCs-0618 [scRNA]; Homo sapiens; RNA-Seq
Using single cell 3' Library and Gel Bead Kit V3.1 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074), the cell suspension (300-600 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. In short, cells at different time points throughout chemical reprogramming process were harvested and resuspended at 1 x 106 cells per milliliter in 1 x PBS with 0.04% BSA. About 1 x 104 cells were added to each channel, and the target cell will be recovered was estimated to be about 5 x 103 cells. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53 ℃ for 45 min, followed by 85 ℃ for 5 min, and hold at 4 ℃. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200. According to the manufacture's introduction, Single-cell RNA-seq libraries were constructed using Single Cell 3' Library and Gel Bead Kit V3.1 The libraries were finally sequenced using an IlluminaNovaseq6000 sequencer with a sequencing depth of at least 1 x 105 reads per cell with pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).