title: NC week 15 rep 2
organism: Mus musculus
description: Protocols: Hearts were minced and incubated in collagenase type II (Worthington) at 37oC before filtering through 40mm strainers. Cells were resuspended in red cell lysis buffer, followed by dead cell removal and two times wash with FACS buffer (2% FBS, 2mM EDTA in PBS, pH 7.4) before the acquisition. For CM nuclei extraction, strainers were washed with FACS buffer to collect CMs in a 50-ml Falcon tube. After 2 rounds of centrifugation (500g x 5 min), CM nuclei were isolated by swelling CMs in a hypotonic solution (91 mM NaCl, 5.3 mM KCl, 0.5 mM MgCl2, 0.293 mM CaCl2, 10 mM glucose, 10 mM HEPES at pH 7.4 and 0.05% Triton-X-100 supplemented with 40 U/ul RNasin® Ribonuclease Inhibitor (Promega Corporation)) before triturating cells using a 30-gauge hypodermic needle to mechanically fragment cells. Nuclei were then isolated by filtration through a 40 µm cell strainer to exclude large debris. Finally, nuclei suspension was centrifuged at (500g x 5 min) before staining with DAPI, followed by FACS for isolation of nuclei. FACS sorted live (DAPI-) TIP cells and CM nuclei were counted, and loaded on to the 10x Chromium microfluidics chip (3-prime v3.1). TIP and CM nuclei were FACS-sorted and subsequently pooled at specific proportions (50% TIP (without CD31+ cells), 20% CD31+ cells and 30% CM nuclei). Isolation of single cells was performed in two batches on separate days, with samples from each treatment group split across days to mitigate batch effects. Cells/nuclei from 2 mice were pooled together after FACS isolation and considered as one replicate and 2 replicates per condition were run for sequencing. FACS-sorted TIP cells and CM nuclei from each heart were manually counted using a hemocytometer and were mixed) and used for single cell transcriptomic library preparation on the Chromium controller (10X Genomics). Approximately 6000 total cells/nuclei (per condition) were loaded into each channel and processed using the Chromium Single Cell 3' v3.1 reagent kit (10X Genomics). Following capture and lysis, we synthesized cDNA and amplified for 12 cycles as per manufacturer's protocol (10X Genomics). The amplified cDNA was used to construct Illumina sequencing libraries that were each sequenced on one lane of an Illumina NovaSeq 6000 to an approximate depth of 30,000 reads per cell. Isolation of single cells was performed in two batches on separate days, with samples from each treatment group split across days to mitigate batch effects. 10x Chromium 3-prime v3.1 protocol 10x Chromium 3-prime v3.1 protocol