title: GSM6499540: Cow 2, bulk RNA sequencing, visceral adipose tissue (VAT); Bos taurus; RNA-Seq
library_construction_protocol: 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. Total RNA from adipose tissue was extracted using the RNeasy Lipid Tissue Mini kit (Qiagen, Cat. No. 74804) and RNA quality was determined using RNA Screen Tape (Agilent). Samples with RNA integrity number (RIN) greater than 7 were used in the subsequent steps. Messenger RNA purification, RNA fragmentation, double stranded cDNA, and adaptor ligation were generated using llumina Stranded mRNA Prep kit according to the manufacture's protocol (Illumina, Cat. No. 20040534). PCR enriched libraries were quantified using the Quant-iT PicoGreen™ dsDNA Assay Kit (Invitrogen, Cat. No. P11496) and equimolar indexed libraries were pooled. Pooled libraries were checked using the Agilent Tapestation 2200 and quantified by qPCR. The libraries were then diluted to 250 pM and spiked with 1% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 flow cell, paired-end reads (2 × 50 bp) targeting at least 30 million reads per sample.