GSM6189429: Cardiac Leukocytes, LOY BMT, TAC, scRNAseq; Mus musculus; RNA-Seq
Hearts were flushed with 15 ml of cold PBS from apex and excised. Right ventricles were removed, and left ventricles were minced and digested in collagenase I (450 U/ml), collagenase XI (125 U/ml), hyaluronidase (450 U/ml), and DNase I (60 U/ml) (Sigma-Aldrich, Cat. #C0130, C7657, H3506, and D4513, respectively) at 900 rpm at 37°C for 30 minutes using ThermoMixer® C (Eppendorf). Hearts were subsequently homogenized through a FisherbrandTM Cell Strainers. After dead cell staining with Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat. #L34957), single cell suspension of heart tissue was incubated with fluorochrome-conjugated antibodies for 20 minutes at room temperature. Viable BMT-derived cardiac immune (DAPI-CD45+RFP+) cells were FACS-isolated (n = 3 per condition, pooled samples after FACS). Single cell suspensions of ~8,000 cells per sample were then used to generate single cell RNA sequencing libraries (Chromium Single Cell v3.1 3' Reagent Kit, 10x Genomics), and nucleotide reads were generated by paired-end next-generation sequencing (P2 2x100 Sequencing Kit, NextSeq 2000 Sequencer, Illumina). Library preparation and sequencing was performed according to validated standard operating procedures established by the University of Virginia Genome Analysis and Technology Core, RRID: SCR_018883.