title: GSM6500444: Young, replicate 4, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Inguinal subcutaneous adipose was carefully dissociated from female C57BL/6N mice. Excised inguinal subcutaneous adipose was rinsed twice thoroughly with Dulbecco's Phosphate-Buffered Saline (DPBS, supplemented with 1% Penicillin-Streptomycin Solution), and then the hair stuck to the adipose tissue was removed carefully. The minced adipose tissues were digested with collagenase D (2mg/ml; Roche) and collagenase type I (2mg/ml; Gibco) in DMEM (Hyclone, USA) for 45 min at 37°C incubator shaker. DNase-I (Sigma, USA) was added at 15min and 30min during digestion. The digestive mixture was quenched with DMEM supplemented with 10% FBS and filtered via 100μm nylon filter, then centrifuged at 400×g for 5 min. Supernatant were discarded and the cell pellet was resuspended with red blood cell lysis buffer (Solarbio, China) for 3min on the ice. The cell lysate was quenched with DMEM supplemented with 10% FBS and centrifuged at 400×g for 5 min. DPBS supplemented with 0.4% bovine serum albumin (Gibco,USA) was used to resuspend the cell pellet. Finally, the cell suspension was filtered through a 40μm filter for 10×Genomics single-cell RNA sequencing. Library was performed according to the manufacturer's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, Stromal vascular fractions (SVFs) were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina TruSeq R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.