GSM9557051: scRNASeq_Subject3; Homo sapiens; RNA-Seq
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 4 women with PCOS. 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. Selected cells were subjected to first-strand cDNA synthesis initiated by oligo dT primer (SMART-Seq ICELL8 CDS), followed by template switching with template switching oligo (SMART-Seq ICELL8 oligonucleotide) for 2nd strand cDNA synthesis, prior to unbiased amplification of full-length cDNA. Synthesized full-length cDNA was tagmented by TDE1 (Illumina Tagment DNA enzyme) and amplified using forward (i5) and reverse (i7) indexing primers. Each single nucleus was indexed by a unique combination of 1 of 72 forward and 1 of 72 reverse indexing primers allowing for downstream identification. Collected cDNA was purified twice using a 1:1 proportion of AMPure XP beads (Beckman, A63880), further amplified according to manufacturer's instructions and purified again at a 1:1 proportion of AMPure XP beads. The resultant sequencing-ready cDNA library was assessed for concentration by fluorometer (Qubit) and quality by electrophoresis (Agilent Bioanalyzer high sensitivity DNA chips) respectively. Libraries were sequenced with Illumina NovaSeq X Plus.