GSM6499544: Cow 1, visceral adipose tissue (VAT), snRNA-seq; Bos taurus; RNA-Seq
Visceral adipose tissue (VAT) was collected from greater omentum and subcutaneous adipose tissue (SAT) from the right flank of the same three lactating Holstein dairy cows in a local abattoir. Samples were snap frozen in liquid nitrogen, transported to the laboratory, and stored at -80°C until further processing. Nuclei isolation from bovine VAT and SAT was adapted from a previous protocol with brain tissue (Krishnaswami et al., 2016). Briefly, 500 mg samples of frozen VAT or SAT were washed with sterile RNase free cold 1X PBS (10XPBS buffer, pH 7.4, Invitrogen, Cat. No. AM9625 diluted 1:10 with Nuclease-free water, Invitrogen, Cat. No. AM9932), then minced with a scalpel using a petri dish on ice. Samples were homogenized in a precooled 15 mL glass dounce homogenizer (10 strokes with pestle A followed by 15 strokes with pestle B) with 1.5 mL of homogenization buffer composed by 5 mM MgCl2 (Invitrogen, Cat. No. AM9530G), 10 mM Tris Buffer (pH 8.0) (Invitrogen, Cat. No. AM9855G), 25 mM KCl (Invitrogen, Cat. No. AM9640G), 250 mM sucrose (Sigma-Aldrich, Cat. No. S0389-500g), 1X protease inhibitor (Roche, Cat. No. 11836170001), 1 µM DL-Dithiothreitol solution (DTT, Sigma-Aldrich, Cat. No. 646563-10X), 0.4 U/µL Ribolock RNase Inhibitor (40 U/μL) (ThermoScientific, Cat. No. EO0381), 0.2 U/µL Superasin (20 U/µL) (Invitrogen, Cat. No. AM2696), and 0.1% Triton X-100 (Sigma-Aldrich, Cat. No. T8787-100ML). Samples were then strained with pre-wet 100 µm and 40 µm filters into a 50 mL conical tube. Next, each sample was transferred to two 1.5 mL pre-chilled microcentrifuge RNase free tubes and centrifuged at 500 × g, 4°C for 5 min. Supernatant was pipetted off leaving approximately 50 µL, which was resuspended in 500 µL of 1% BSA-PBS (Bovine serum albumin, Sigma-Aldrich, Cat. No. A7030-10G) including 0.2 U/μL of Ribolock RNase Inhibitors. Before proceeding with fluorescence activated cell sorting (FACS), a subsample of nuclei was used to assess the overall quality of the nuclei by staining with trypan blue and visualized by phase-contrast light microscopy. To enable sorting of nuclei, we immune-stained the nuclei sample with propidium iodide (PI) (Invitrogen, Cat. No. V13241, 10 ug/mL) in a 1:100 dilution, leaving approximately 20-30 µL of sample to be used as unstained control. Each sample was transferred into a pre-coated 5 mL polystyrene flow cytometry tube and sorted using a 100 µM nozzle in a BD FACSAria II Cell Sorter (BD Biosciences, Franklin Lakes, NJ, USA). Sorting strategy included doublet discrimination and selection of intact nuclei by sub-gating on PI stanning. PI+ nuclei was sorted directly into a 1.5 mL microcentrifuge tube containing 20 µL of 1% BSA-PBS and 0.2 U/μL of Ribolock RNase Inhibitors. After nuclei isolation, samples were centrifuged at 100 × g, 4°C for 6 min, and the supernatant was pipetted off leaving approximately 50 µL of resuspended nuclei sample. Single nuclei suspensions were subjected to final nuclei counting on an automated cell counter (Countess 2, Life Technologies Inc., Carlsbad, CA, USA) and diluted to a concentration of 700-1,000 nuclei/µL. 3' single nuclei libraries were generated following manufacturer's user guide: 10X Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index). Final library quality was resolved using DS 5000 HS assay kit using Tape Station 4200 (Agilent Technologies, Santa Clara, CA, USA). The libraries were quantified using Qubit dsDNA HS assay kit on Qubit Fluorometer version 2.0 (Life Technologies Inc., Carlsbad, CA, USA). Pooled libraries were subjected to 150 bp paired-end sequencing according to the manufacturer's protocol (Illumina NovaSeq 6000). Bcl2fastq2 Conversion Software (Illumina) was used to generate de-multiplexed Fastq files and the CellRanger Pipeline (10X Genomics) was used to align reads and generate count matrices