GSM9384863: Pancreatic islet cells, ATF6a cKO, Normal diet-fed, Batch 1; Mus musculus; RNA-Seq
Islets were isolated by collagenase digestion through the bile duct using collagenase P with incubation at 37°C for 30 min, followed by density gradient centrifugation with Histopaque solutions. Isolated islets from two mice were hand-picked and pooled for each sample, filtered through a cell strainer, dissociated into single cells with accutase, and adjusted to ~1,000 cells/µL in PBS. Library construction was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 5' Reagent Kit v2 and Chromium Controller, 10x Genomics). Briefly, single-cell resuspensions were loaded onto the Chromium Controller for Gel Bead-in-Emulsion (GEM) generation. Within each GEM, reverse transcription attached a cell-specific barcode and Unique Molecular Identifier (UMI) to cDNA generated from poly-A RNA. After GEM breakage, barcoded cDNA was recovered, cleaned using Silane DynaBeads, and amplified. The amplified cDNA was then fragmented, end-repaired, A-tailed, ligated with sequencing adaptors, and indexed by PCR to generate libraries. Libraries were sequenced on an Illumina NovaSeq 6000 using 150-bp paired-end reads.