GSM9045875: Single nuclei, Acute Day 3 post injection of iECM,  biol rep 1 and 2; Rattus norvegicus; RNA-Seq
Nuclei from cardiac tissue was isolated as mentioned previously. In brief, flash frozen LV free wall samples were resuspended in nuclei lysis buffer (Millipore Sigma, Nuclei EZ prep, NUC101), 0.2 U/L RNase inhibitor (Enzymatic Y9240L) and finely chopped with scissors. Samples were homogenized with a 2 mL dounce grinder (KIMBLE) with the lysates filtered through a 100 µm, 50 µm, 20 µm strainers (CellTrics filters), respectively. The samples were centrifuged at 1000g for 5 min at 4C to pellet nuclei. The nuclear pellet was resuspended in a sucrose gradient buffer  and centrifuged at 1000g at 4ºC for 10 minutes. The pellet was then washed with a nuclei storage buffer containing 10 μg ml−1 4′,6-diamidino-2-phenylindole DAPI and centrifuged at 500 g and 4ºC for 5 minutes, with the pellet resuspending in 200 mL of 2% BSA in 1x PBS. Nuclei were then counted on a hemocytometer and volume adjusted to 1000 nuclei/µL for loading. snRNA-seq was performed with a microfluidic droplet-based technique provided by 10X Genomics (Chromium Next GEM Single Cell 3' HT v3.1). Quality control for cDNA and libraries were performed on Aligent TapeStation, and library concentrations were determined via Qubit HS DNA kit. Paired-end sequencing was run on a NovaSeq6000 instrument. Demultiplexing of sequenced samples were mapped to a rat reference genome (Rnor6.0, including introns). Redundant unique molecular identifiers (UMIs) were eliminated via the Cell Ranger 7.1.0 or Space Ranger v1.3.1 from 10X Genomics. snRNA-seq was performed with a microfluidic droplet-based technique provided by 10X Genomics (Chromium Next GEM Single Cell 3' HT v3.1). Quality control for cDNA and libraries were performed on Aligent TapeStation, and library concentrations were determined via Qubit HS DNA kit. Paired-end sequencing was run on a NovaSeq6000 or NovaSeq X Plus instrument. Demultiplexing of sequenced samples were mapped to a rat reference genome (Rnor6.0, including introns). Redundant unique molecular identifiers (UMIs) were eliminated via the Cell Ranger 7.1.0 pipeline or Space ranger v1.3.1 from 10X Genomics.