GSM7596043: NCD; scRNAseq; Mus musculus; RNA-Seq
Samples were washed by PBS to remove the remaining blood cells and then cut into small pieces.These small pieces were digested with Type II collagenase at 1 mg ml-1 for 30 min at 37 ℃ while shaking at 120 rpm. The samples were prepared into single-cell suspensions, Cell counts and cell viability were measured with the Countess® II Automated Cell Counter with cell activity above 80%, and cell concentrations were adjusted to the ideal concentration of 1000 /μL. scRNA-seq libraries were generated using the Chromium Single Cell 3' Library and Gel Bead Kit V3 (10X Genomics, 1000075), Chromium Single Cell B Chip kit (10X Genomics, 1000074), and Chromium i7 Multiplex Kit (10X Genomics, 120262). Cells were resuspended in reverse transcription enzyme mix and loaded onto the Chromium Single Cell B Chip to generate gel beads-in-emulsion of 6,000 cells per channel. Reverse transcription was performed on an S1000TM Touch Thermal Cycler (Bio-Rad). The cDNA was generated and then amplified, and quality was assessed using an Agilent 4200. ScRNA-seq libraries were constructed using Single Cell 3' Library and Gel Bead Kit V3 and finally sequenced using an Illumina NovaSeq 6000 instrument with a sequencing depth of at least 50,000 reads per cell with paired-end 150-bp reading strategy.