GSM8462861: resHif1aKO, control 1, day 3 MI, snRNAseq; Mus musculus; RNA-Seq
Hearts were perfused with PBS, weighed, minced, and digested for 45 minutes at 37⁰C in DMEM (Gibco 11965-084) containing 4500 U/ml Collagenase IV (Millipore Sigma C5138), 2400 U/ml Hyaluronidase I (Millipore Sigma H3506), and 6000 U/ml DNAse I (Millipore Sigma D4527). Enzymes were deactivated with HBSS containing 2% FBS and 0.2% BSA and filtered through 40-micron strainers. Cells were incubated with ACK lysis buffer (Gibco A10492-01) for 5 minutes at room temperature. Cells were washed with DMEM and resuspended in 100 µl of FACS buffer (PBS containing 2% FBS and 2mM EDTA). Cells were incubated in a 1:200 dilution of fluorescence conjugated monoclonal antibodies for 30 minutes at 4⁰C. Samples were washed in FACS buffer and resuspended in a final volume of 250 µl FACS buffer. Flow cytometry and sorting was performed using a BD FACS melody. For sorting of monocytes, macrophages, and dendritic cells in macHif1aKO mice for single cell RNA sequencing, antibodies used were: CD45 PerCP/Cy5.5 (BioLegend 103132), Ly6g FITC (BioLegend 127606), Ly6c APC (BioLegend 128016), DAPI (BD Pharmingen 564907), and CD64 PE/Cy7 (BioLegend 139313). For sorting of monocytes, macrophages, and dendrtic cells in Arg1ZsGr, antibodies used were: CD45 PerCP/Cy5.5(BioLegend 103132), CD11b BV421 (BioLegend 101235), Ly6g APC/Cy7 (BioLegend 127623), Ly6c BV510 (BioLegend 128033), CD64 PE/Cy7 (BioLegend 139313). Endogenous ZsGreen signal was used to sort ZsGr- and ZsGr+ cells. For sorting of monocytes and macrophages in Arg1tdT-CreERT2 Csf1rlsl-DTR-mCherry mice 3 days after I/R, antibodies used were: CD45 PerCP/Cy5.5 (BioLegend 103132), CD11b BV785 (BioLegend 101243), Ly6g APC/Cy7 (BioLegend 127623), Ly6c BV510 (BioLegend 128033), CD64 PE/Cy7 (BioLegend 139313). For single nucleus RNA sequencing of control and resHif1aKO, hearts from 6 Control and 6 resHif1aKO mice were perfused with PBS and minced, and nuclei were isolated using the 10X Genomics Nuclei Isolation Kit with RNase Inhibitor (PN-1000494). The resulting single nuclei preparations were pooled based on experimental condition and were stained with a 1:200 dilution of DRAQ5 (ThermoFisher Scientific 65-0880-96). DRAQ5+ nuclei were sorted into 300 µl of Wash Buffer with RNase Inhibitor. For monocytes, macrophages, and dendritic cells from macHif1a KO and control mice: Single cell RNA sequencing libraries were generated using the 10X Genomics 5'v1 platform. For ZsGr- and ZsGr+ monocytes, macrophages, and dendritic cells from Arg1ZsGr mice 2 and 30 days after myocardial infarction: Single cell RNA sequencing libraries were generated using the 10X Genomics 5'v1 platform. For ZsGr- and ZsGr+ monocytes, macrophages, and dendritic cells from Control>ZsGr and Hif1a>ZsGr mice 5 days after syngeneic heart transplant: Libraries were generated using the 10x 3'v3.1 platform. For monocytes and macrophages from control and Arg1DTR mice sorted 3 days after myocardial infarction: libraries were generated using the 10x 3'v3.1 platform. For nuclei isolated from control and resHif1aKO mice 3 days after myocardial infarction: 2 libraries were generated using the 10X Genomics 5'v2 platform.