GSM6333286: SAT SVC,  SARS-CoV-2-infected, Participant 2, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
On the day of bariatric surgery, approximately 2-3g each of subcutaneous abdominal (SAT) and omental visceral adipose (VAT) tissue was harvested intraoperatively and immediately processed. Tissue was subject to collagenase digestion for separation of mature adipocytes (MA) and SVC, and for differentiation of preadipocytes as previously described in PMID: 28836202 with details in supplemental methods. SVC from SAT and VAT was left untreated or infected with SARS-CoV-2 as described above. After 24hpi, cells were collected, washed, and diluted at a density of 1x103 cells per µl in cold DMEM (Life technologies; 11885-092) media supplemented with 10% FBS (Corning, MT35016CV). The gel beads-in-emulsion (GEM) single cell 3' kit v3.1 dual index kit (10X Genomics) was used following manufacturer's recommendations with slight modifications. Briefly, SVC from SAT and VAT was left untreated or infected with SARS-CoV-2 as described above. After 24hpi, cells were collected, washed, and diluted at a density of 1x103 cells per µl in cold DMEM (Life technologies; 11885-092) media supplemented with 10% FBS (Corning, MT35016CV). 10,000 cells per lane were loaded onto a Chromium Controller in the BSC per manufacturer's instructions. Following GEM creation, samples were transferred into PCR tube strips prior to transferring into a PTC-200 thermocycler (MJ Research) for RT. The RT parameters were the following: 45 minutes at 53oC, followed by 5 minutes at 85oC, then 15 minutes at 60oC and finally samples were kept at 4oC. Barcoded cDNA was removed to BSL2 and sequencing libraries were prepared per manufacturer's recommendation, with a TapeStation 4200 (Agilent) used for quality control. Libraries were pooled for a final concentration of 10nM and sequenced on a NovaSeq S2 (Ilumina) at the Chan Zuckerberg Biohub (San Francisco).