GSM8136444: Tcf21-MCM baseline; scRNA-seq; Mus musculus; RNA-Seq
Single cells were obtained by flow cytometry of live nucleated cells from cardiac tissue digests. Single-cell droplet libraries were generated according to manufacturers' instructions (Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual index), PN-1000268, 10X Genomics; user guide CG000315). Samples had a greater than 70% viability. About 16,000 cells were loaded to recover 10,000 cells. Briefly, gel emulsion beads (GEM) were generated by combining gel beads, master mix containing the cells and partitioning oil and loaded onto a Chromium Next GEM Chip G at a low dilution. Poly-adenylated mRNA from the cell lysate contained in every GEM was reverse transcribed to cDNA adding an Illumina R1 primer sequence, unique molecular identifier (UMI) and the 10X barcode. The barcoded first-strand cDNA was then purified with MyoOne Silane DynaBeads (20000048, 10x Genomics) and amplified using 14 PCR cycles. The full-length barcoded cDNA was then enzymatically fragmented and size selected using SPRIselect (B23317, Beckman Coulter) and adaptor ligated. The cDNA was then amplified for library construction adding an Illumina R2 primer sequence, paired-end constructs with the P5 and P7 sequences used for Illumina amplification and sample indexes were added.