title: Illumina HiSeq 4000 sequencing; Myocardial infarction time series single cell RNA-Seq
design: Myocardial infarction time series single cell RNA-Seq
library_construction_protocol: Adult murine hearts(ventricles) were collected at homeostasis or different time points post_MI.  Heart ventricles were dissected, mechanically cut into small pieces with a scalpel and subjected to two 15min digestions with 263U/ml collagenase type II (Worthington Biochemical Corporation, LS004177), under magnetic agitation at 250rpm at 37C. Cell suspensions were passed through a 40 μm cell strainer to remove cardiomyocytes, small fragment of undigested tissue and collected on ice in a conical 50ml tube containing with 2%FCS (VWR Seradigm, 97068-085). Cells were spun at 300g for 5 min and pre-warmed red cell lysis buffer (Sigma-Aldrich, R7757) was added for 1 min to remove blood cells. After washing, cell pellets were resuspended in 200 μl of Dead Cell Removal MicroBeads (Miltenyi Biotec, 130-090-101) and incubated 10 min at room temperature in the dark. The mix of cells and beads was diluted in binding buffer (Miltenyi Biotec, 130-090-101) and an AutoMACS Separator was used to deplete dead cells bound to the beads.  After centrifugation at 400g for 5 min, cells were resuspended in 2%FCS+2mM EDTA and stained with 2µM DRAQ5 (eBioscience, 65-0880-92) followed by 3µM Propidium Iodine (Sigma-Aldrich, P4864) after 5min. Single live nucleated cells were sorted on a BD AriaII with 130µm nozzle to minimize pressure over cells (12psi). 16-32,000 events were sorted in 0.04%BSA, centrifuged for 8min at 400g, resuspended and diluted to 320-400cells/µl for scRNAseq (10x Genomics).   Cells capture, lysis and RNA extraction were performed using the 10x V2 Chromium technology. About 12000 cells were loaded in each lane of the 10xChromium chip. Barcoding and library preparation were performed with the 10x Chromium system using version 2 chemistry, according to the manufacturer's protocol (10x Genomics, #CG00052). cDNA was synthesized and amplified (12 cycles) as per manufacturer's protocol  and amplified to construct an Illumina sequencing library. cDNA and libraries were checked for quality on an Agilent 4200 TapeStation, quantified by KAPA qPCR.
attributes.tag: Experimental Factor: time
attributes.value: 0
attributes.tag: Experimental Factor: genotype
attributes.value: Wt1^Cre; ZsGreen^f/+
attributes.tag: Experimental Factor: injury
attributes.value: none