GSM8746885: D7_scRNA; Mus musculus; RNA-Seq
For scRNA-seq sample collection, we scraped all cells with different induction phenotypes at each time point, aiming to analyze all cell types involved in the induction process. Cells were washed in ice-cold PBS and dissociated using SeekMate Tissue Dissociation Kits (SeekGene). DNase I treatment was applied if needed. Cell viability was assessed using a Fluorescence Cell Analyzer (Countstar® Rigel, S2), after removing erythrocytes and filtering out debris and dead cells. Fresh cells were resuspended in RPMI1640 with 2% FBS at 1×106 cells/mL. Single-cell RNA-Seq libraries were prepared using the SeekOne® Digital Droplet Single Cell 3' Kit (SeekGene). Cells were mixed with reverse transcription reagents and loaded into SeekOne® chip S3 for droplet generation. Reverse transcription was performed at 42°C for 90 minutes, followed by inactivation at 85°C for 5 minutes. cDNA was purified, amplified, fragmented, end-repaired, and ligated to sequencing adaptors. Indexed PCR was performed to amplify the 3' polyA regions, including Cell Barcodes and Unique Molecular Indices (UMIs). Libraries were cleaned, quantified, and sequenced on an Illumina NovaSeq 6000 or DNBSEQ-T7 platform.