GSM8338577: Myocardial infarction s/p retroviral PHF7 intramyocardial injection [snRNA-seq]; Mus musculus; RNA-Seq
Heart nuclei were extracted using a modified version of a previously described protocol in ref (PMID: 34382019). Hearts were dissected, snap-frozen, and stored at -80C. For each conditions n=1-3 hearts were pooled together. Hearts were thawed for 5 min on ice and the minced with scissors in 1 ml cold lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NonidetTM P40, 1 mM DTT, 1 U/μl RNase inhibitor in Nuclease-Free Water). After 2 min, 4 ml of cold lysis buffer was added, and hearts were lysed for 5 min at 4 °C. 10 ml of cold wash buffer (PBS, BSA 2%, and 0.2 U/μl RNase inhibitor from Roche) was added, and the lysate was dounced with 20 strokes of a loose pestle. The homogenate was filtered with 70 μm and 40 μm cell strainers. Nuclei were pelleted by centrifugation for 5 min at 500 × g at 4 °C. Nuclei were resuspended in 0.5 ml of wash buffer. Nuclei were resuspended in 0.5 ml of wash buffer and 2 μl of anti-Nuclear Pore Complex conjugated antibody (Biolegend, 682203) to stain all nuclei. After 15 min, nuclei were pelleted by centrifugation for 5 min at 500 × g at 4 °C. Then the nuclei were resuspended in 300 μl of wash buffer and filtered with 30μm cell strainers. More than 200,000 nuclei were FACS sorted with a BD FACSAria III and the BD FACSDIVA and FlowJo v10.8.1 software. Nuclei were pelleted by centrifugation for 10 min at 500 × g at 4 °C and then resuspended in 200 μl of ATAC lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% NonidetTM P40, 0.001% Digitonin, 1% BSA, 1 mM DTT, 1U/ul RNase inhibitor in Nuclease-Free Water). After 2 min, 1.3 ml of ATAC wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1 mM DTT, 1% BSA, 1U/μl RNase inhibitor in Nuclease-Free Water) was added. Nuclei were pelleted by centrifugation for 10 min at 500 × g at 4 °C and resuspended in 20 μl of diluted nuclei buffer from 10X genomics. Library was performed according to the manufacter's instructions (Next GEM single-cell Multiome ATAC+ Gene expression kit). Briefly, Post GEM-incubation Cleanup using DynaBeads MyOne Silane Beads was followed by Pre-Amplification PCR (72 °C for 5 min, 98 °C for 3 min, cycled 7 × 98 °C for 20 s, 63 °C for 30 s, 72 °C for 1 min). After cleanup with SPRIselect Reagent Kit, ATAC library was generated (98 °C for 45 s, cycled 8 × 98 °C for 20 s, 67 °C for 30 s, 72 °C for 20 s) and purified with SPRIselect Reagent Kit. The fragment size estimation of the resulting ATAC libraries was assessed with Agilent TapeStation D1000 HS (Agilent) and quantified using the QubitTM double stranded DNA (dsDNA) High-Sensitivity HS assay (ThermoFisher Scientific). SnATAC-seq libraries were then sequenced by pair with a HighOutput flowcel using an Illumina Nextseq 500 with the following mode: Read 1N - 63 cycles, i7 index – 8 cycles, i5 index - 24 cycles *(8 dark cycles and 16 cycles on i5), Read 2N - 63 cycles. SnRNA-seq libraries were constructed by performing the following steps: cDNA Amplification, SPRIselect cleanup, Fragmentation, End reair and A tailing, SPRIselect cleanup, and Adaptor ligation, SPRIselect cleanup, Samplex index PCR and SPRIselect cleanup size selection. The fragment size estimation of the resulting libraries was assessed with Agilent TapeStation D1000 (Agilent) and quantified using the QubitTM double stranded DNA (dsDNA) High-Sensitivity HS assay (ThermoFisher Scientific).