GSM5025922: S23, Hyper-organoids origin from ENR-organoids, p1; Mus musculus; RNA-Seq
The sampling details for the scRNA-seq experiments are shown in Supplementary information, Table S3. To isolate single cells, Hyper-organoids and ENR-organoids were incubated with TrypLE Express at 37 °C for 20 min, after which live cells were counted using a hemocytometer. On average, ~3000 captured individual cells per sample were subjected to 10× Genomics single-cell isolation and RNA-seq following the manufacturer's recommendations. Using single cell 3′ Library and Gel Bead Kit V3 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074), the cell suspension (300–600 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10× Genomics) 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. Captured cells were lysed and the released RNA was 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 °C for 5 min, and hold at 4 °C. The cDNA was generated and then  amplified, and quality assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing). According to the manufacture's introduction, scRNA-seq libraries were constructed using Single Cell 3′ Library and Gel Bead Kit V3. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).