GSM8415341: Cultured DFAT cells, iWAT, scRNA seq; Mus musculus; RNA-Seq
Adherent cells were gently washed 3 times with PBSto remove residual culture medium and debris. An appropriate amount of 0.25% Trypsin-EDTA(Gibco, Cat#25200-056) was added to ensure complete coverage of the cells, which were then incubated at 37°C for 3-5 minutes. Cell morphology changes were observed under a microscope, and when the cells became round and began to detach from the culture plate, the plate was gently shaken to facilitate cell detachment. Complete culture medium containing serum was added to terminate the dissociation process. Complete culture medium containing serum was added to terminate the dissociation process. The cell suspension was centrifuged at 450g for 10 minutes to obtain a cell pellet, which was then resuspended in sorting buffer supplemented with 2% FBS. 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.