title: GSM7100387: Inguinal White Adipose, Aged, 3 Days Cold; Mus musculus; RNA-Seq
library_construction_protocol: As previously described (Merrick et al 2019, Wang et al 2019), iWAT tissue was dissected, minced gently and digested with Collagenase Type I (1.5 units/ml; Worthington) and Dispase II (2.4 units/ml; Roche) in DMEM/F12 containing 1% fatty acid–free bovine serum albumin (Gold Biotechnology) in a gentleMACS dissociator (Miltenyi Biotec) on program “37 MR ATDK-1.” The digestion was quenched with DMEM/F12 containing 10% FBS, and the dissociated cells were passed through a 100 uM filter and spun at 400 xg for 4 mins. The pellet was resuspended in red blood cell lysis buffer (Biolegend), incubated for 4 mins at RT, then quenched with DMEM/F12 containing 10% serum. Cells were passed through a 70 uM filter, spun, resuspended, then passed through a final 40 uM filter, spun at 400 xg for 4 minutes and plated or underwent further processing for FACS. Cells were flow sorted to isolate live (DAPI(−)) cells and remove debris. We enriched non-immune cells by sorting out CD45+ cells. Next-generation sequencing libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell 3' Reagent kit v3.1 per manufacturer's instructions. Libraries were uniquely indexed using the Chromium Single Index Kit T Set A, pooled, and sequenced on an Illumina NovaSeq 6000 sequencer in a paired-end, dual indexing run by the CHOP Center for Applied Genomics at the University of Pennsylvania. Sequencing for each library targeted 20,000 mean reads per cell.