GSM8283893: hCiPSC_24 wpt,scRNA-seq; Homo sapiens; RNA-Seq
CiPSC-derived islets were dissociated into single cells using Accutase, collected and resuspended at 1 × 106 cells per mL in 1× PBS with 0.04% BSA. Using Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, 1000268) and Chromium Next GEM Chip G Single Cell Kit (10x Genomics, 1000120), the cell suspension (700–1200 living cells per μL 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 brief, CiPSC-derived islets were dissociated into single cells using Accutase, collected and resuspended at 1 × 106 cells per mL in 1× PBS with 0.04% BSA. About 8 × 103 cells were added to each channel, and the target cell will be recovered was estimated to be about 5 × 103 cells. Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual gel beads in emulsion (GEMs). Reverse transcription was performed on a T100 Touch Thermal Cycler (Bio-Rad) at 53 °C for 45 min, followed by 85 °C for 5 min, and then held at 4 °C. The cDNA was generated and then amplified, and quality-assessed using the Qsep1 system. According to the manufacturer's instructions, scRNA-seq libraries were constructed using the Library Construction Kit . The libraries were quality-assessed using the Qsep1 system . The libraries were finally sequenced using the Illumina NovaSeq 6000 sequencer with a sequencing depth of at least 2 × 105 reads per cell with a paired-end 150 bp (PE150) reading strategy.