GSM7091457: I/R, replicate 2, scRNAseq; Mus musculus; RNA-Seq
Mice hearts were harvested and frozen after sacrificing them. Frozen tissue samples were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 ml of ice-cold nuclei EZ lysis buffer. The sample was then incubated in ice for 5 min with 3 ml of cold EZ lysis buffer. Nuclei were centrifuged at 500 g for 5 min at 4°C, washed with 5 ml ice-cold EZ lysis buffer, and incubated on ice for 5 min. After centrifugation, the nuclear pellet was washed with 5 ml nuclei suspension buffer (NSB; consisting of 1% PBS, 0.01% BSA, and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)). Isolated nuclei were resuspended in 2 ml NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. A final concentration of 1,000 nuclei per μl was used for loading onto a 10x channel. Beads with a unique molecular identifier (UMI) and cell barcodes were loaded close to saturation such that each cell was paired with a bead in a gel beads-in-emulsion. After exposure to the cell lysis buffer, polyadenylated RNA molecules were hybridized into the beads. Beads were retrieved in a single tube for reverse transcription. For complementary deoxyribonucleic acid (cDNA) synthesis, each cDNA molecule was tagged on the 5' end (that is, the 3' end of a messenger RNA transcript) with UMI and a cell label indicating its cell of origin. Briefly, 10× beads were subjected to second-strand cDNA synthesis, adaptor ligation, and universal amplification. Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3' end of the transcripts linked to the cell barcode and UMI. All remaining procedures, including library construction, were performed according to the standard manufacturer's protocol (Chromium Single Cell 3ʹ v3). Sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and Qubit high- sensitivity DNA Assay (Thermo Fisher Scientific). The libraries were sequenced on NovaSeq6000 (Illumina) using 2 × 150 chemistry.