GSM8828571: Day 6 EPS-embryoids-1; Mus musculus; RNA-Seq
EPS-derived embryoids were digested by 0.05% trypsin/TripLE into single cells, then washed and resuspended in 1X PBS (calcium and magnesium free) containing 2% FBS.  Cell suspensions were filtered through a 40 μm cell strainer.Cell viability was determined by Count Star, and the density of living cells were adjusted to 300-600 living cells per microliter. Cell suspension was loaded onto the Chromium single cell controller (10x Genomics). Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10x Genomics) and Chromium Single Cell B Chip Kit (10x Genomics) were used to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. In short, single cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were added to each channel, and the target cells were recovered (about 3,000 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°C for 45 min, followed by 85°Cfor 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200. Single-cell RNA-seq libraries were constructed using Single Cell 3' Library and Gel Bead Kit V3.1 according to the manufacture's introduction. The libraries were finally sequenced using an IlluminaNovaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy.