scRNA-Seq from stroma vascular fraction of abdominal and gluteofemoral subcutaneous white adipose tissue
SVF was isolated from ABD and GF-adipose tissue biopsies by collagenase digestion and centrifugation, from 5 healthy women (38yo ±4.4; 31.8kg/m-2 ±3.12; WHR 0.86 ±0.05). After isolation, cells were counted with a Countess II automated cell counter (Thermofisher Scientific). Single cell suspension (56K/mL) was distributed into eight wells of a 384-well source plate (Takara Bio USA, San Jose, CA) and dispensed onto a iCELL8 350v Chip (Takara Bio USA) using an iCELL8 MultiSample NanoDispenser (Takara Bio USA). We distributed the ABD and GF-SVF of each subject on the same chip. The method used was described in [6]. In brief, mRNA from single cells were isolated, converted to full-length cDNA prior to unbiased amplification of 3' and 5' ends using SMART-Seq® ICELL8® Application Kit (Takara Bio, USA). Multiplexed sequencing libraries were generated from cDNA using the Illumina Nextera XT protocol and 150bp paired-end sequencing was performed on an Novogene HiSeqX instrument. Overall design: SVF was isolated from ABD and GF-adipose tissue biopsies tissue was digested with collagenase (type 1, 1 mg/ml in HBSS) for 45min at 37ºC with shaking (100 rpm). The mixture was centrifuged at 500g for 5 minutes and floating adipocytes were discarded. Cell pellets were treated with erythrocyte lysis buffer (Invitrogen) for 5 min at room temperature. After discarding the supernatant, cells were repelleted with centrifugation, resupend in PBS/EDTA and the mixture was passed through 100 and 40 micron mesh filters. After centrifugation, the cells were resuspended in PBS/10% FBS/1%EDTA and counted with a Countess II automated cell counter (Thermofisher Scientific). SVF were isolated from 5 healthy women (38yo ±4.4; 31.8kg/m-2 ±3.12; WHR 0.86 ±0.05).  Single cell suspension (56K/mL) was distributed into eight wells of a 384-well source plate (Takara Bio USA, San Jose, CA) and dispensed onto a iCELL8 350v Chip (Takara Bio USA) using an iCELL8 MultiSample NanoDispenser (Takara Bio USA). We distributed the ABD and GF-SVF of each subject on the same chip.