title: GSM8296047: islets, Isl1CKO,6; Mus musculus; RNA-Seq
library_construction_protocol: P9 mice were sacrificed, and pancreas was perfused by Collagenase I (1mg/ml) with Actinomycin D (25µg/ml) and dissected. Tissue was enzymatically digested with 1mg/ml collagenase and incubated at 37°C for 10-12mins. Digested tissue was washed with 1% FBS in Hank's salt solution 3x. Dissociation of the tissue with 0,05% trypsin/0.53 mM EDTA supplemented with Actinomycin D (25 µg/ ml) at 37°C for 5 mins helped further release endocrine cells. FACS buffer (2% FBS and 10 mM EGTA in PBS) was used to stop digestion and to wash cells 1x. Cell suspension was filtered using 40µm nylon mesh and  tdTomato+ cells were sorted on  FACSAria™ Fusion flow cytometer. According to manufacturer protocol, sorted single cell suspension was loaded to Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10 × Genomics, Pleasanton, CA) to prepare cDNA libraries. Individual cells were encapsulated into droplets with barcoded gel beads, unique molecular identifiers, and poly(dT) sequences. Reversely transcribed cDNA was amplified (1-14 cycles), fragmented and ligated to sequencing adaptors. Purification of cDNA and size selection of fragment was done with SPRISelect magnetic beads. Libraries were pair-end sequenced Illumina NovaSeq 6000 SP Reagent Kit.