GSM6833321: Library 15 - Peritoneal macs High-fat/low-fat diet: 3hrs; Mus musculus; RNA-Seq
Cells were washed once with cold PBS after 3hrs of stimulation and lifted into suspension by incubating at 37C for 5 minutes with Accutase, which resulted in cell viability typically >85%. Cells were centrifuged at 4C, 400g for 5 minutes, and resuspended in PBS + 2% FBS for the Rhapsody scRNAseq assay. Cells were hash-tagged with anti-CD45-hashtags (BD Rhapsody # 633793) and loaded onto the cartridge following manufacturer's instructions (BD Rhapsody # 633771), with the following modifications, which helped ensure sufficient cell viability for the subsequent steps: Incubation with hashtags was performed for 30mins on ice, instead of 20mins at room temperature; only two washes were performed after hashtag incubation to minimize cell loss. Each cartridge was then loaded with a total of ~36k cells across 12 hash-tagged samples (~3k cells/sample). Libraries were prepared according to manufacturer's instructions (BD Rhapsody # 633771) and sequenced 2x100 on Novaseq 6000.