title: GSM6152893: STZ treated, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: The mice were anesthetized and the bile duct were injected with 0.5 mg/ml collagenase P dissolved in cold Hanks' balanced salt solution. The expanded pancreatic tissue was digested at 37°C for 11 min and then stopped using cold Hank's Balanced Salt Solution (HBSS) with 10% fetal bovine serum (FBS). The islets were separated using a density gradient composed of layers of HBSS and Histopaque (11191; Sigma Aldrich). After two rounds of manual selection, the islets were dispersed into single cells with TrypLE (Thermo Scientific) for 15 min in a 37°C water bath and then resuspended in Dulbecco phosphate-buffered saline (containing 2% FBS). GFP+ cells were sorted using flow cytometry and confirmed to have >80% viability using Trypan blue staining. Single cells were processed to produce gel bead-in-emulsion (GEM) using a Chromium™ Single Cell Platform (10x Genomics) with a Chromium™ Single Cell 3' GEM, Library & Gel Bead Kit v3 and a Chromium™ Chip B Single Cell Kit, following the manufacturer's protocol. Thereafter, the beads were retrieved into a single tube for reverse transcription, and the cDNAs were tagged at the 5' ends (that is, the 3' ends of mRNAs were tagged) with a unique molecular identifier (UMI) and a label indicating each molecule's cell of origin (CG000206 Rev D). The libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a 2100 Bioanalyzer system (Agilent) with a Qubit High Sensitivity DNA Assay Kit (Thermo Fisher Scientific). The libraries were sequenced (2x150 bp) on a NovaSeq 6000 system (Illumina).