title: GSM8652329: RT1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: SMA-mTmG mice from one year of age were euthanized at either room temperature (RT, 22C) or thermal neutral temperatures (TN, 30C). Adipose tissue samples, specifically inguinal fat depots, were carefully excised from experimental mice. A pair of inguinal adipose depots from each mouse were minced and placed in 10 ml of isolation buffer (0.1 M HEPES, 0.12 M NaCl, 50 mM KCl, 5 mM D-glucose, 1.5% BSA, 1 mM CaCl2) containing collagenase type I (10,000 units). The tissue was incubated at 37ºC with gentle agitation for approximately 1 hour. Following digestion, Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 Ham (DMEM/F12) (Sigma, cat. no. D8900 and D6421) was added to the mixture, which was then strained through a 100 μm cell strainer. The samples were centrifuged at 200xg for 10 minutes, and the supernatant was discarded. The resulting pellet was resuspended in 10 ml of erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 minutes, followed by the addition of DMEM/F12 growth media supplemented with 10% fetal bovine serum (FBS). The suspension was strained through a 40 μm cell strainer and centrifuged again at 200xg for 5 minutes. The cell pellet was resuspended in growth media and submitted to the Cornell Transcriptional Regulation and Expression (TREx) Facility at Cornell University for further processing. The RNA sequencing (RNAseq) project was managed by the Transcriptional Regulation and Expression (TREx) Facility at Cornell University. Stromal vascular fractions (SVF) from the inguinal fat depot were collected and immediately processed following established protocols to ensure cell viability and RNA integrity. For cell viability, SVF cells were counted using an Invitrogen Countess Automated Cell Counter, and viability was assessed using ReadyCount™ Stains (Invitrogen, Cat. A49905), ensuring >85% viable cells for downstream processing. SVF cells were resuspended at a concentration of 1,000 cells/μL in PBS containing 0.04% bovine serum albumin (BSA) and loaded onto the 10x Genomics Chromium Controller for single-cell partitioning. Isolated RNA underwent comprehensive quality control analysis at the TREx Facility. Concentration determination was performed using a Nanodrop spectrophotometer, chemical purity was assessed, and RNA integrity was evaluated with an Agilent Fragment Analyzer. These steps ensured high-quality input for downstream processes. For library preparation, Poly(A)+ RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). A total of 750 ng of RNA per sample was used to prepare directional RNAseq libraries with the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs). Library quantification was conducted using a Qubit dsDNA HS Kit (Thermo Fisher Scientific) to ensure accurate input for sequencing. Adipose tissue was further processed for single-cell RNA sequencing (scRNAseq). Single-cell suspensions were prepared by enzymatic digestion using collagenase type II and filtration through a 70 µm cell strainer to obtain single cells. Approximately 10,000 cells were loaded onto a Chromium Chip G to generate gel beads in emulsion (GEMs) using the 10x Genomics Chromium Controller. Reverse transcription was performed within the GEMs to produce barcoded cDNA molecules, ensuring unique molecular identifiers (UMIs) for each transcript. After GEM generation, the GEMs were broken, and the cDNA was recovered, purified, and amplified through polymerase chain reaction (PCR). The amplified cDNA was assessed for size distribution and concentration using an Agilent Bioanalyzer 2100. For library preparation, the amplified cDNA was enzymatically fragmented, end-repaired, and A-tailed. Sample indexing adapters were ligated to the cDNA fragments, and indexed libraries were further amplified via PCR. Final libraries underwent quality control, where size distribution was evaluated using an Agilent Bioanalyzer, and library concentration was quantified using a Qubit Fluorometer (Thermo Fisher Scientific, Cat. #Q33240). cDNA libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics). The quality of these libraries was assessed using an Agilent Bioanalyzer 2100. Finally, the libraries were pooled and sequenced on an Illumina NovaSeqX platform, targeting a read depth of 10,000 reads per cell. No modifications were made to the standard 10x Genomics protocol.