GSM8184324: Heart, Control, ScRNAseq, rep1; Mus musculus; RNA-Seq
Cell suspensions were prepared by mechanically cutting the hearts into small pieces with a scalpel and incubating with an enzymatic digestion mix (see protocol) for 30 min, under magnetic stirring at 250rpm, 37ºC. The cell suspension was passed through a 40 mm cell strainer to remove cardiomyocytes, small fragment of undigested tissue. Cells were spun at 300g for 5 min and pre-warmed red cell lysis buffer was added for 1 min to remove blood cells. After washing, cell pellets were resuspended in 200 mL Dead Cell Removal MicroBeads and incubated 10 min at room temperature in the dark. The mix of cells and beads was diluted in binding buffer and pass through the magnatic columns to remove dead cells. After centrifugation, cells were resuspended in and stained with  3 mM DAPI. Single live nucleated cells were sorted on a BD AriaII with 130mm nozzle to minimize pressure over cells and sorted in 0.04%BSA, centrifuged for 8min at 400g, resuspended and diluted to 1000cells/ul for scRNaseq (10x Genomics). Cells were then further processed according to manufacturer's (10X) instructions. The scRNA-seq libraries were prepared using the 10x Chromium Single Cell 3′ Solution. Quality control of all cDNA samples was performed with a Bioanalyzer 2100 (Agilent Technologies) and libraries were quantified with the Qubit dsDNA HS kit. Gene expression libraries were sequenced on an Illumina NextSeq 1000 using 20,000 reads per cell. Libraries were performed according to the manufacter's instructions (single cell 3' v3.1 protocol, dual index, 10x Genomics).