GSM4192749: Total CD45+ cells from obese group; Mus musculus; RNA-Seq
Perigonadal adipose was isolated and washed with ice-cold PBS. Adipose was then minced to 2–3 mm pieces, added 4 mL of enzymatic digestion mix (1 mg/mL bovine-serum albumin, 0.77 mg/mL Liberase, 15.8 mU Hyaluronidase, 25 mU DNAse1 in Hanks' Balanced Salt solution) and transferred to gentleMACS C-tubes. Tissue was then dissociated using the gentleMACS Octo Dissociator, program name: mr_adipose_01 , ran 3 times. Suspensions were subsequently filtered with a 100 µm cell strainer, washed with ice-cold PBS and stained for cell-sorting. Live CD45+ cells were sorted out using BD FACSAriaIII into DMEM containing 10% FBS. After centrifugation, cells were resuspended in final concentration of ~500 cells/µL in 1X HBSS containing 0.04% BSA to minimize cell loss and aggregation. ~12,000 cells are partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) to achieve single cell resolution for a maximum recovery of 10,000 individual cells per sample. Utilizing the v2 Chromium™ Single Cell 3' Library Kit and Chromium instrument (10x Genomics), poly-adenylated mRNA from an individual cell is tagged with a unique 16bp 10x barcode and 10bp Unique Molecular Identifier. Full length cDNA is then amplified to generate sufficient mass for library construction. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size (~400bp) for library construction. The final library is sequence ready and contains four unique sample indexes. The concentration of the 10x single cell library will be accurately determined through qPCR (Kapa Biosystems) to produce cluster counts appropriate for the HiSeq4000 platform (Illumina). 26x98nt read lengths are generated targeting 50,000 reads per cell.
GEO Accession
GSM4192749