GSM9149535: Primary white adipose SVF cells (P0); Mus musculus; RNA-Seq
After reaching confluency (in 4 days), cells were washed with PBS and lifted with Trypsin-EDTA for 3 minutes. WAT and BAT progenitor suspensions were then washed in isolation media, counted and live cells processed for single-cell sequencing Single-cell RNA sequencing was conducted by the University of Wisconsin-Madison Biotechnology Center's Gene Expression Center Core Facility using the 10x Genomics Chromium Single Cell 3ʹ v3.1 platform. Single-cell suspensions were loaded onto the Chromium Next GEM Chip, where cells were partitioned into Gel Bead-In-EMulsions (GEMs) for reverse transcription (RT). Post GEM-RT, cDNA was recovered and purified using DynaBeads MyOne Silane beads (Lot# 160221), followed by amplification through 12 cycles of PCR. The amplified cDNA underwent cleanup with SPRIselect beads (0.6X, Lot# 18198500) and quality was checked using an Agilent HS DNA chip. For library construction, cDNA was fragmented, end-repaired, and A-tailed, with double-sided size selection using SPRIselect beads (0.6x and 0.8x, Lot# 18198500). Adapter ligation was performed, followed by post-ligation cleanup with SPRIselect beads (0.8x). Index PCR was carried out with 7 cycles using the Chromium Dual Index TT Set A (Lot# 160185). The final library was cleaned using SPRIselect beads (0.6x and 0.8x), recovering 35 μL in EB buffer. Library concentration was determined using a Qubit Fluorometer with Qubit dsDNA HS reagents. Final quality control was performed using an Agilent TapeStation with D1000 ScreenTape. Sequencing was initially conducted on a MiSeq Nano, followed by final sequencing on an SP flow cell.