GSM6310691: iBAT, C57, cold 4 days, Linpos, rep 2; Mus musculus; RNA-Seq
Interscapular brown adipose tissues (iBAT) were surgically removed and processed for stromal vascular cell (SVC) isolation. Tissues of 3-4 mice were pooled after digestion. iBAT pads were washed with PBS, minced, and digested with 2 mg/mL type 2 collagenase (Worthington Biochemical Co.) in Hanks' balanced salt's solution (HBSS; Sigma-Aldrich) containing 4 mM sodium bicarbonate, 10 mM HEPES (pH 7.4, Gibco; Sigma-Aldrich) and 0.5% fatty acid free bovine serum albumin (FF-BSA; Gemini Bioproducts, West Sacramento, CA) for 30 minutes at 37°C. Ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 10 mM and samples were incubated for an additional 5 minutes to promote complete dissociation of SVCs. Digested cell samples from the same treatment group were combined and filtered through 100 μm and 40 μm sterile cell strainers. The samples were washed with PBS buffer containing 1 mM EDTA, 2.5 mM HEPES, and 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals Inc) (FACS buffer) and centrifuged at 500 x g for 10 minutes at 4°C. After removing the supernatant, pelleted stromal vascular cells were incubated in red blood cell lysis buffer containing 14 mM sodium bicarbonate, 0.154 M ammonium chloride, and 0.1 mM EDTA for 5 minutes at room temperature, then passed through a 100 μm sterile cell strainer. Cells were collected by centrifugation at 500 x g for 10 minutes at 4°C. Cellular debris was removed by density gradient centrifugation using Debris Removal Solution (Miltenyi Biotec). Cells recovered were resuspended in PBS containing 0.5% FF-BSA. iBAT SVCs were separated into lineage-marker positive (Lin+) and lineage-marker negative (Lin-) cell fractions using magnetic bead cell separation (MACS). SVCs were labeled using the mouse Lineage Cell Depletion kit (Miltenyi Biotec) according to the manufacturer's instructions. This kit contains anti-CD5, anti-CD11b, anti-CD45R (B220), anti-Gr-1 (Ly-6G/C), anti-7-4, and anti-Ter-119 antibodies. Labeled samples were passed onto MS columns (Miltneyi Biotec) on the OctoMACS separator (Miltenyi Biotec); the flow through was collected as the Lin- cell fraction and bound Lin+ cells were eluted from the column. Samples were centrifuged at 500 x g for 10 minutes at 4°C and pelleted cells were resuspended in PBS with 0.04% FF-BSA. Cells were counted and diluted to a concentration of 1,000 cells/uL. Single-cell libraries for all samples were prepared using the 10X Genomics Chromium Single Cell 3' Reagent Kit v2, apart from ADRB1 KO and WT room temperature libraries which were prepared with Reagent Kit v3. Following MACS, single-cell suspensions were loaded onto the Single Cell Chip A. An estimated 10,000 cells were loaded per lane for an expected recovery of ~6,000 cells per library. The assembled chip was placed on a 10X Genomics Chromium Controller Instrument (10X Genomics) to generate single-cell gel beads in emulsion (GEMs). Single-cell RNA-seq libraries were prepared according to the manufacturer's instructions. Libraries were quantified using the Kappa PCR kit (Kappa Biosystems) and sequenced with the Illumina NextSeq500 using high output 75-cycle kits with the following read length configuration: v2 libraries 26 bp read1, 8 bp I7 index, and 58 bp read2, v3 libraries 28 bp read1, 8 bp I7 index, and 56 bp read2.