title: GSM9024552: eWAT_SVF_18HFD, scRNA seq; Mus musculus; RNA-Seq
library_name: GSM9024552
library_construction_protocol: iWAT and eWAT were dissected, washed twice with HBSS, then minced and digested in 1 mg/ml collagenase type I (Sigma-Aldrich, C0130) dissolved in digestion buffer (0.1 M HEPES-Na, 120 mM NaCl, 50 mM KCl, 5 mM D-glucose, 1 mM CaCl₂, 1.5% BSA) for 45 min at 37°C with continuous shaking (150 rpm). Digestion was quenched by adding an equal volume of growth medium (Advanced DMEM/F-12 [Gibco, 12634028] supplemented with 10% FBS, 1% GlutaMAX [Thermo, 35050061], 1% MEM NEAA [Thermo, 11140050], and 1% penicillin-streptomycin [Gibco, 15140122]). The suspension was sequentially filtered through 100-μm and 40-μm cell strainers (Fisher Scientific), with centrifugation (450 g for 10 min) between steps. Erythrocyte lysis was performed by resuspending the pellet in red blood cell lysis buffer (5 min, 37°C), followed by neutralization with growth medium and final filtration with the 40-μm strainer. Isolated cells were stained with 7-AAD at a concentration of 5 μg/mL and subjected to FACS sorting. Number of cells were quantified with a Countstar Rigel S5 Automated Cell Counter. Approximately 20,000 cells were applied to the Chromium microfluidic chips with 3' v3 chemistry and barcoded with a 10x Chromium Controller using the Single-Cell 3′ Reagent Kit v3.1 (10x Genomics). The following program was used for GEM-Reverse Transcription: 53℃ for 45 min, 85℃ for 5 min, and held at 4℃. The resulting emulsions were lysed for cDNA isolation and purification with the Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Scientific) and amplified with PCR. cDNA quality was assessed with an Agilent 2100 Bioanalyzer. cDNA amplicon size was optimized by enzymatic fragmentation and size selection. P5, P7, i7, and i5 sample indexes and TruSeq Read 2 (read 2 primer sequence) were added by end repair, A-tailing, adaptor ligation, and PCR, respectively. Final libraries containing the P5 and P7 primers were sequenced by an Illumina NovaSeq 6000 sequencer with 150 bp paired-end reads, aiming for a coverage of approximately 50,000 raw reads per cell.