GSM9228264: Pancreatic islets, Slc7a11-MacWT; Mus musculus; RNA-Seq
Single cell solutions were obtained from pancreatic islets isolated from 6-8 C57BL/6J males as previously described. Immune cells were sort-purified as live DAPI-Epcam-CD45+ cells with a FACS ARIA III cell sorter (BD Biosciences) using FACS Diva software (BD Biosciences) and lysed for mRNA extraction. Sorted cells were then centrifuged at 300 rcf for 5 minutes and resuspended in PBS containing 0.04% of Ultrapure BSA (Invitrogen Cat# AM2616). Cells were loaded on a Chromium Controller (10x Genomics) with a target output of 3000-4000 cells per condition and RNA-seq libraries were prepared with the Chromium Single Cell 3' Reagents Kit v3 Library protocol (10x Genomics) according to manufacturer's guidelines at the CurieCoreTech Next Generation Sequencing (ICGex, Paris, France). After individual qualification and quantification, the libraries were then equimolarly pooled and quantified using a qPCR method (KAPA library quantification kit, Roche). The sequencing was carried out on an Illumina Novaseq 6000 instrument, using paired-end 28x91bp as sequencing mode, at the Curie Genomic Platform (Paris, France). The sequencing configuration was set to reach an average of 50,000 reads per cell.