NextSeq 500 sequencing; Single-cell RNA-seq of the human Lineage– cells derived from the adipose stromal vascular fraction across depots
Single-cell RNA-seq of the human Lineage– cells derived from the adipose stromal vascular fraction across depots
SVF cells isolated following the dissection protocol, were stained for 30 min on ice with CD31 and CD45 antibodies according to the dilution in Supp. Table 4 or according to Miltenyi manufacturer protocol, then washed twice with 1%Albumin in DPBS prior to enriching for the SVF Lin– fraction using FACS or MACS (Supp. Table 3). The sorted cells were washed once and resuspended to the concentration of 800 cells/ul in 1%Albumin in DPBS prior to loading them on the 10x Chromium device according to the manufacturer protocol. 2-3 cm3 biopsies from Subcutaneous (SC), Omentum (OM), Perirenal (PR), Mesocolic (MC) and Gallbladder-associated (GB) adipose tissues were washed in PBS to remove excess blood, weighted and finely minced using scissors. Minced adipose tissue was incubated with 0.28 U/ml of liberase TM (Roche #05401119001) in DPBS with calcium and magnesium (Gibco #14040091) for 60 min at 37 °C under agitation. Vigorous shaking was performed after 45 min of incubation to increase the yield of recovered SVF cells. The digested tissue was mixed with an equal volume of 1% human albumin (CSL Behring) in DPBS -/- (Gibco #14190094) to stop the lysis. Following a 5-min centrifugation at 400 g at room temperature, floating lipids and mature adipocytes were discarded by aspiration and the resuspended SVF pellet was sequentially filtered through 100-μm and 40-μm cell strainers to ensure a single cell preparation. To lyse red blood cells, pelleted SVF was resuspended in VersaLyse solution (Beckman Coulter #A09777) according to the manufacturer's recommendations and washed once with 1% albumin solution. Obtained red blood cell-free SVF suspension was then stained to isolate the lineage (CD45, CD31) negative fraction through FACS or MACS. Immediately after the sample collection protocol, the samples were loaded into the Chromium Single Cell Gene Expression Solution (10x Genomics), following the manufacturer's recommendations targeting a recovery of 4000 to 5000 cells per run. scRNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3′ v2 or v3 Reagent Kit. Immediately after the sample collection protocol, the samples were loaded into the Chromium Single Cell Gene Expression Solution (10x Genomics), following the manufacturer's recommendations targeting a recovery of 4000 to 5000 cells per run. scRNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3′ v2 or v3 Reagent Kit.