title: GSM7113248: M_JC-WT-6wk-1-lib1; Mus musculus; RNA-Seq
library_construction_protocol: Freshly isolated hearts from 16-week-old WT, Dsg2mut/mut and Dsg2mut/mut x Ccr2-/- mice (n=3 per group) were minced on ice with a razor blade, transferred to a 15 mL conical tube containing 3mL DMEM with 170uL collagenase IV (250U/mL final concentration), 35uL DNAse1 (60U/mL), and 75uL hyaluronidase (60U/mL), and incubated at 37 °C for 45 min with agitation. Thereafter, the digestion reaction was quenched with 5 mL of HBB buffer (2% FBS and 0.2% BSA in HBSS), passed through 40-µm filters into a 50 mL conical tube and transferred back into a 15 mL conical tube to obtain tighter pellets. Samples were then spun down at 4°C, 1200rpm for 5 min and the supernatant was discarded. Pellets were resuspended in 1 mL ACK Lysis buffer (Gibco A10492-01)    and incubated at room temperature for 5 min, followed by the addition of 9 mL DMEM and centrifugation (4°C, 5 min, 1200 rpm). Supernatant was discarded and the pellets were resuspended in 2 mL FACS buffer (2% FBS and 2mM EDTA in calcium/magnesium free PBS); centrifugation was repeated in above conditions and supernatant aspirated. The TotalSeq A 277 panel (BioLegend) antibody cocktail was resuspended in 100 µL of FACS buffer and added to each sample. The combined 100 µL were used to resuspend the pellet with the addition of 1 µL of DRAQ5 (5mM solution Thermo Fisher Scientific, 564907) and incubated on ice for 30 min. Solution was washed 3x with FACS buffer following same centrifugation as above and then resuspended in 300 µL of FACS buffer and 1 uL DAPI (BD Biosciences, 564907) and filtered into filter top FACS tubes. First singlets were gated and subsequent DRAQ5+/DAPI- events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS) – collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells/µL. Cells were counted on a hemocytometer before proceeding with the 10x protocol.    Sample preparation for single nuclei RNA-seq (snRNA-seq): Frozen hearts from 16-week-old WT, Dsg2mut/mut and Dsg2mut/mut x Ccr2-/- mice (n=3 per group) were minced with a razor blade and transferred into a 5 mL Dounce homogenizer containing 1–2 mL chilled lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% NP-40 in nuclease-free water). Samples were homogenized gently using five passes without rotation, and then incubated on ice for 15 min. Lysate was gently passed through a 40-μm filter into 50-mL conical tube, followed by rinsing the filter once with 1 ml lysis buffer and transfer of lysate to a new 15-mL conical tube. Nuclei were then centrifuged at 500g for 5 min at 4°C, followed by resuspension in 1 mL Nuclei Wash Buffer (2% BSA and 0.2 U /µL RNase inhibitor in 1× PBS) and filtered through a 20-μm pluristrainer into a fresh 15-mL conical tube. Centrifugation was repeated according to the above parameters. Supernatant was then removed, and    nuclei were resuspended in 300 µL Nuclei Wash Buffer and transferred to a 5-mL tube for flow sorting. Then, 1 µl DRAQ5 (5 mM solution; Thermo Fisher, cat. no. 62251) was added, mixed gently and allowed to incubate for 5 min before sorting. DRAQ5+ nuclei were sorted into Nuclei Wash Buffer. Recovered nuclei were centrifuged again under the above parameters and were gently resuspended in Nuclei Wash Buffer to a target concentration of 1,000 nuclei/µL. Nuclei were counted on a hemocytometer. Chromium Single Cell 5ʹ Reagent v1.1 kit from 10X Genomics and Chromium Single Cell 3ʹ Reagent v1.1 kit from 10X Genomics