GSM5018059: atac_iBATpos; Mus musculus; ATAC-seq
For isolation of adipose SVF cells, fat tissues were dissected into ice-cold PBS and minced with scissors in a sterile 5 ml tube containing 4 ml of either iBAT digestion buffer (1X HBSS, 3.5% BSA, and 2 mg/ml collagenase type II) and incubated at 37°C for 1 hr under agitation or WAT digestion buffer (1X DPBS, 1% BSA, 2.5 mg/ml dispase and 4 mg/ml collagenase D) for 40 min. The digestion mixture was passed through a 100 μm cell strainer into a 50 ml tube. Digestion was stopped by adding 15 ml PBS containing 2% fetal bovine serum (FBS; Gibco, 10270106) and centrifuged at 500 g for 5 min at room temperature. The supernatant was aspirated, and red blood cells were lysed by incubating the SVF pellet with 2 ml RBC lysis buffer (Invitrogen, 00433357). The number and viability of cells in the suspension were determined using Trypan Blue stain according to the manufacturer's recommendations. Following separation by FACS,cells were loaded using Chromium Single Cell 3' v2 Reagent Kit, and libraries were run on the Illumina NextSeq 550 Sequencing System as 150-bp paired-end reads. To isolate nuclei for scATAC-seq assay, cells were lysed for 4 min on ice according to 10x genomics protocol (CG000169 Rev D). ScATAC-seq libraries were prepared using the 10x Genomics platform with the Chromium Single Cell ATAC Library & Gel Bead Kit as recommended by the manufacturer and sequenced on the Illumina NextSeq 550 Sequencing System as 150-bp paired-end reads.