GSM7336853: Day2, scRNAseq; Mus musculus; RNA-Seq
Heart samples from neonatal C57BL/6 mice were dissociated into single-cell suspensions. The tissues were minced with a razor blade, and then digested in digestion buffer with 0.75% Collagenase/Dispase. The cell suspension was filtered with cell strainers, and centrifuged. Subsequently, the cell pellet was resuspended with PBS and treated with 1X Red Blood Cell Lysis Solution to remove red blood cells. Trypan blue was used to check the viability of cells. 10x Library Preparation and Sequencing Beads with unique molecular identifiers (UMI) and cell barcodes are loaded to near saturation so that each cell is paired with a bead in a gel bead emulsion (GEM). After exposure to cell lysis buffer, polyadenylated RNA molecules hybridize to the beads. Beads were collected in one tube for reverse transcription. In cDNA synthesis, each cDNA molecule is labeled at the 5' end (ie, the 3' end of the messenger RNA transcript), and the UMI and cell tag indicate its cellular origin. Briefly, 10x beads are followed by second-strand cDNA synthesis, adapter ligation, and universal amplification. Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products, enriched for 3' ends of transcripts linked to cellular barcodes and UMIs. All remaining steps, including library construction, were performed according to the standard manufacturer's protocol (CG000206 RevD, 10x Genomics). Sequencing libraries were quantified using the High Sensitivity DNA Chip (Agilent) and Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific) on the Bioanalyzer 2100.