GSM4663165: Lin- iAdFASNKO; Mus musculus; RNA-Seq
Fat pads were cut and minced in digestion buffer and incubated in a 37°C water bath for 45 min. The digested tissue was passed through a 100-μm-pore-size cell strainer and centrifuged at 600g for 10 min. The pelleted cells were collected as the SVF, and red blood cells were lysed by incubation with red blood cell lysis buffer for 5 min. The SVF was centrifuged again, and the cell pellet was resuspended in 1 ml of PBS. This final cell suspension solution was passed through a 40um cell strainer to discard debris and get single cells for Drop-Seq application. Single cell RNA-seq libraries were prepared using the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (10X Genomics) according to the manufacturer's protocol. The SVF of iWAT from control and iAdFASNKO mice were isolated and subjected to single-cell RNA-seq analysis. To better resolve the relatively low numbers of immune cells present in the SVF, the cells were isolated and fractionated into lineage marker-positive (Lin+), mostly immune cells, and all other stromal cells (Lin-) using magnetic bead cell sorting. Thus, four different libraries were prepared from an estimated approximately 6,000 cells per condition (Control and iAdFASNKO, Lin+ and Lin-), and were subjected to sequencing using Illumina NovaSeq 6000.
GEO Accession
GSM4663165