title: GSM9101242: Subcutaneous adipose tissue, Cow 10, Control; Bos taurus; RNA-Seq
library_construction_protocol: Approximately 500 mg of each adipose tissue sample was washed in cold, sterile, RNase-free 1X PBS (CAT# AM9625, Invitrogen) diluted in nuclease-free water (CAT# AM9932, Invitrogen), then minced using a scalpel in a petri dish on ice in 0.5 mL lysis Buffer comprised of nuclease-free water with 3 mmol/L MgCl2 (CAT# AM9530G, Invitrogen), 10 mmol/L Tris Buffer, pH 8 (CAT# AM9855G, Invitrogen), 25 mmol/L KCl (CAT# AM9640G, Invitrogen), 250 mmol/L sucrose (CAT# S0389-500G, Sigma-Aldrich), 0.1% Triton X-100 (CAT# T8787, Sigma-Aldrich), and 100 µmol/L DTT (CAT# 646563, Sigma-Aldrich). After mincing, samples were transferred and homogenized in a pre-cooled 15 mL glass dounce homogenizer (CAT# 357544, Duran Wheaton Kimble) by performing 10 strokes with Pestel A and 15 strokes with Pestel B on ice. Samples were then strained through a 40 µm filter (CAT# 22363547, ThermoFisher Scientific), pre-wet with ± 200 µL of Nuclei Isolation Medium (NIM; composed of Lysis Buffer without Triton and DTT) into a 50 mL conical tube. The strained homogenates were then transferred into pre-cooled, sterile, RNase-free microcentrifuge tubes and centrifuged at 500 x g for 5 min at 4 ºC. The resulting supernatant was discarded, leaving ± 50 µL in the tube. The pellet was then resuspended in 500 µL of nuclei resuspension buffer containing 1% BSA (CAT# 9048-46-8, ThermoFisher Scientific), and 0.2 U/µL RNase inhibitor (CAT# 11836170001, Roche) in DPBS (CAT# 14190-144, Gibco). The samples were then centrifuged at 300 x g for 5 min at 4 ºC, after which the supernatant was removed, leaving ± 50 µL in the tube. The sample was gently resuspended in the remaining volume and 5 µL was removed for counting nuclei using Trypan Blue (CAT# 15250-061, Gibco), while the remaining sample was utilized for sequencing. Nuclei libraries, targeting 6,000 - 10,000 nuclei, were prepared using the 10X Genomics Chromium Next GEM Single Cell 3' Reagent Kits (10X Genomics). Library quality was assessed via DS 5000 HS assay kit using Tape Station 4200 (Agilent Technologies, Santa Clara, CA, United States) and quantified using Qubit dsDNA HS assay kit on Qubit Fluorometer version 2.0 (Life Technologies Inc., Carlsbad, CA, United States). Nuclei libraries were sequenced via 150bp paired-end sequencing using an Illumina NovaSeq 6000 (Illumina). FASTQ files were generated using the Bcl2fastq2 Conversion Software (Illumina), and the CellRanger Pipeline (10X Genomics) was utilized for alignment and generating count matrices.