title: GSM7791492: F6_CytoID_pos; Mus musculus; RNA-Seq
library_construction_protocol: After 15 days of culture, single cells were isolated from organoids via incubation for 5 minutes with 1X Dispase (354235; Corning) in HBSS followed by incubation for 1 hour at 37°C with shaking at 1000 RPM in 0.25% Trypsin-EDTA (25-510; Genesee Scientific) supplemented with 10 μM Y27632. Cells were then forced through a cell strainer (70 μm) into a tube containing 4 ml 250 μg/ml soybean trypsin inhibitor (17975-209; Gibco) in PBS.  Cells were pelleted then resuspended in complete 'mouse' keratinocyte serum-free medium (KSFM): KSFM without calcium chloride (10725-018, Gibco, Waltham, MA, USA) with recombinant epidermal growth factor (1 ng/ml), bovine pituitary extract, (50 mg/ml), 1% penicillin/streptomycin (15140-122; Gibco, and 0.018 mM CaCl2 (349610025; Acros Organics). Single cell suspension was enriched for live cells using Dead Cell Removal kit (130-090-101; Miltenyi Biotec) according to the manufacturer's protocol. Single cell droplets were generated with chromium single-cell controller using Chromium Next GEM Single Cell 3' Kit v3.1 (1000121; 10x Genomics). 5000–7000 cells were collected to make cDNA at the single cell level. Full-length cDNA with unique molecular identifiers (UMIs) was synthesized via reverse-transcription in the droplet. After PCR amplification and purification, cDNA was fragmented to around 270 bp and Illumina adapters with index were ligated to fragmented cDNA. After PCR, purification, and size selection, single cell RNA libraries were 450 bp in length and sequenced on Illumina sequencer at R1 = 28 bp, R2 = 91 bp.