GSM7886255: Sorted FBs TAC Il1b-KO_Rep3; Mus musculus; RNA-Seq
Isolation from mouse hearts were performed using two methodologies: to isolate non-cardiomyocytes (non-CM) cells from the ventricles using Langendorff perfusion (#1) and to isolate all nuclei from the ventricles using a Langendorff-free protocol (#2). For #1, after a proper level of anesthesia was induced, a thoracotomy was performed and the mouse heart was isolated, cannulated, and perfused with perfusion buffer (120.4 mM NaCl, 14.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-HEPES, 4.6 mM NaHCO3, 30 mM taurine, 10 mM 2,3-butanedione monoxime, and 5.5 mM glucose, pH 7.0) in a Langendorff perfusion system (Radnoti 120108EZ) for 5-10 minutes at 37°C. The cannulated heart was then digested with digestion buffer (perfusion buffer with 300 units/mL collagenase II (Worthington Biochemical) and 50 μM CaCl2) for approximately 10 min at 37°C. At the end of digestion, the atria and great vessels were removed and the ventricular tissue was transferred and gently teased into small pieces in stop buffer (perfusion buffer with 10% fetal bovine serum) at 37°C. After gently pipetting, the cell suspension was passed through a 250 μM strainer in a 50ml conical centrifuge tube and centrifuged at 30xg for 3 minutes at room temperature (RT). The supernatant, which contained most of the non-cardiomyocytes (non-CMs), was divided from the pellet (which contained the CM fraction). The non-CM fraction was centrifuged again at 30xg for 3 minutes at RT and the supernatant retained. The supernatant was filtered with a cell strainer (70 μm) and centrifuged at 400xg for 3 minutes at RT for eliminating debris. The non-CM pellet was resuspended in 1mL cold PBS 0.5% BSA. Cells were counted with trypan blue using a hemocytometer and 15k cells were used for subsequent 10X Genomics Chromium single-cell RNAseq preparation. For single cell ATAC, 500k isolated and purified non-CMs were resuspended in 100uL lysis buffer (Tris-HCl 10mM pH 7.4, NaCl 10mM, MgCl2 3mM, Tween-20 0.1%, P40 0.1%, Digitonin 0.01%, BSA 1% in nuclease-free water), pipetted 10 times, and kept on ice for 5 minutes. Nuclei were washed with 1mL 1X PBS with 1% BSA and centrifuged for 5 minutes at 4 °C. The nuclei pellet was resuspended in 1mL 1X PBS with 1% BSA and filtered with a 10uM strainer (pluriSelect #43-50010-00). Nuclei were counted after DAPI staining using a hemocytometer and 10k non-CM nuclei were used for subsequent 10X Genomics Chromium single-cell ATAC-seq preparation. finally centrifuged at 400xg for 3 minutes in stop buffer at RT and the supernatant was discarded. For #2, after the mouse heart was isolated it was rinsed in a sterile 10cm petri dish containing 1X PBS on ice to remove excess blood and transferred to a 10cm petri dish containing 2ml of ice cold lysis buffer (320 mM sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 2 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8.0, 0.20% triton X-100) and minced into very small pieces. The minced heart tissue in 2ml of lysis buffer was transferred to a “Type A” dounce homogenizer and subjected to 50 strokes. This solution was subsequently transferred to a “Type B” dounce homogenizer and subjected to 3 strokes. The solution was serially passed through 100 μM and 70 μM strainers and then centrifuged at 1,000xg at 4 °C for 8 minutes. The nuclear pellet was resuspended in nuclei resuspension buffer (430 mM sucrose, 70 mM KCl, 2 mM MgCl2, 10 mM Tris-HCl ph 7.2, EGTA 5 mM, 10% glycerol). Nuclei were counted after DAPI staining using a hemocytometer and 15,000 nuclei were used for subsequent 10X Genomics Chromium single-nuclei RNAseq and 10,000 nuclei were used for ATAC-seq. For sc/snRNA-seq library preparation, cells or nuclei were loaded onto the 10X Genomics Chromium instrument according to manufacturer's protocols (Chip G, PN-1000120). All experiments were conducted with version 3.1 NEXT GEM reagents (PN-1000121), using 9 cycles of cDNA amplification for the GEM kit (PN-1000123) and 9 cycles of library amplification for library kit (PN-1000157). Each sample was indexed with a unique sample primer (Single Index Kit T Set A, PN-000213).