title: GSM5699628: SubjectA SVF and Adipocytes; Homo sapiens; RNA-Seq
library_construction_protocol: Adipose tissue biopsies were collected from subcutaneous abdominal adipose tissue. Tissue was digested with collagenase buffer (Type I collagenase in αMEM supplemented with 1% BSA) at 37 °C for 45 min. Adipocytes were collected by centrifugation at 200g for 5 minutes and washed twice with PBS prior to nuclei isolation (see below). After the adipocytes were collected, the SVF was isolated by additional centrifugation at 500g for 5 minutes and washed with RBC lysis buffer. The SVF solution was subsequently filtered through 100μm and 40μm strainer (BD Falcon) and counted with a countess II automated cell counter (Thermofisher). Following a wash, cells were resuspended in 1% BSA- nuclease free-water. Washed adipocytes (~150µL) were homogenized in a glass dounce with 1ml of homogenization buffer (5mM MgCl2, 25mM Tris Buffer pH 8.0, 25mM KCL, 250mM sucrose, 1µm DDT, 1 x protease inhibitor, 0.2 U/µL SUPERase•In RNase Inhibitor (Thermofisher Scientific) in nuclease-free water). Triton-X100 (0.1% v/v) was added to the homogenate prior to incubation on ice for 30 min with regular vortexing. Samples were filtered through a 100µm and 40µm strainer (BD Falcon), centrifuged at 1,000g for 10 min at 4°C, resuspended in 1mL nuclei isolation medium (5mM MgCl2, 25mM Tris Buffer pH 8.0, 25mM KCL, 1 mM EDTA, 0.2 U/µL Ribolock RNAase inhibitor, 1% BSA in nuclease-free water) and centrifuged at 1,000g for 10 min at 4°C. Nuclei was resuspended in 500µL nuclei isolation medium and filtered 10 x with a 25g syringe prior to being counted. Frozen adipose tissue (~100mg) was pulverized under liquid nitrogen and homogenized in 2mL of homogenization buffer (5mM MgCl2, 25mM Tris Buffer pH 8.0, 25mM KCL, 250mM sucrose, 1µm DDT, 1 x protease inhibitor, 0.2 U/µL SUPERase•In RNase Inhibitor (Thermofisher Scientific) in nuclease-free water) with a glasscol homogenizer. The homogenate was incubated for 30 minutes on ice with the addition of Triton-X100 (0.1% v/v) with regular vortexing. Samples were filtered through a 100µm and 40µm strainer (BD Falcon), centrifuged at 2,700g for 10 min at 4°C, resuspended in homogenization buffer and recentrifuged again at 2,700g for 10 min at 4°C. The pellet was then re-suspended in 1mL nuclei isolation medium (5mM MgCl2, 25mM Tris Buffer pH 8.0, 25mM KCL, 1 mM EDTA, 0.2 U/µL Ribolock RNAase inhibitor, 1% BSA in nuclease-free water) and centrifuged at 2,700g for 10 min at 4°C. Following re-suspension in 500µL nuclei isolation medium, sample was filtered 10 x with a 25g syringe prior to being counted. Single-nuclei suspension (40K/mL) or single-cell suspension (28L/mL) was distributed into eight wells of a 384-well source plate (Takara) and dispensed onto a ICELL8® 350v Chip (Takara) using an ICELL8 MultiSample NanoDispenser (MSND, Takara). Following dispense, the chip nanowells were imaged using the ICELL8 Imaging Station to identify nanowells containing a single nucleus/ a live single cell. Only these nanowells were subjected for downstream dispenses. After imaging, the chip was subjected to freeze-thaw to lyse the cells/nuclei, followed by a 3 min incubation at 72oC to denature the RNA. Selected cell/nuclei 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 cell/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 HiSeq 4000.