NextSeq 500 sequencing; scRNA-seq of Mus musculus subcutaneous Lin- cells derived from the subcutaneous adipose stromal vascular fraction
scRNA-seq of Mus musculus subcutaneous Lin- cells derived from the subcutaneous adipose stromal vascular fraction
The experiment was performed using the Chromium System (10x Genomics), following the manufacturer's protocol. SVF was isolated from six 8 week-old mice (3 male, 3 female) as described above. Cells were stained with the anti-mouse antibodies CD31-AF488, CD45-AF488, TER119 AF488 (Biolegend #303110, #304017 and #116215, respectively) and 280k Lin- cells were isolated using a Becton Dickinson FACS Aria II sorter. Cells were washed and resuspended in 250µl FACS buffer (PBS, 2% FBS, 1mM EDTA), targeting the required 1,000 cells/µl concentration, accounting for a 10-20% loss. We pipetted 9.7 µl cell suspension (concentration of 913 cells/µl, ~ 8,800 cells), targeting the recovery of ~ 5,000 cells.  Single-cell RNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3' v2 Reagent Kit. Single-cell RNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3' v2 Reagent Kit.