GSM9361108: LennyiEHM2innervatedheartmuscle5KSakib_H_2_L004; Homo sapiens; RNA-Seq
Frozen iEHMs were used to isolate nuclei, adapted from Sakib et. al. [PMID: 33554149] with modifications. In brief, 500 µL EZ prep nuclei lysis buffer (Sigma, #NUC101) were added to the frozen samples and dounce homogenized for 40-60 strokes (until not big tissue chunks were seen) using plastic pestles in a 1.5 mL DNA low bind tube. Lysates were transferred into a 2 mL DNA low bind tube and additional lysis buffer were supplemented to fill up to 2 mL mark. Samples were incubated for 5 minutes on ice and later centrifuged for 500 xg for 5 minutes at 4 °C. Supernatants were discarded and the nuclei pellets were resuspended into 2 mL lysis buffer, and incubated on ice for 5 minutes. After centrifugation (500 xg, 5 minutes, 4 °C), the pellet was resuspended into Nuclei Suspension buffer, NSB (0.5% OmniPur BSA (Merck Millipore, #2905), 1:200 RNasin® Ribonuclease Inhibitor (Promega, #N2615), 1x cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche, #04693132001), dissolved in 1x PBS (RNAse free, Invitrogen, #AM9624). Filtered through 0.22 µm Syringe Filter Unit (Millipore, #SLGVR33RB)). It was centrifuged again for 500 xg, 5 minutes at 4 °C, and resuspended into 500 µL NSB. The lysate was strained through 40 µM Flowmi® Cell Strainers (Sigma, #BAH136800040) into a FACS tube. 1:500 of 7-AAD (Invitrogen, #00-6993-50) nuclei staining solution was added and nuclei were gated from the debris (Supplementary Figure 8) and sorted with a BD FACSaria III with 85 µM Nozzle, in a 15 mL falcon tube, coated with and containing 1 mL NSB. Around 200,000 nuclei were sorted per sample. Sorted iEHM nuclei were immediately taken to process for single nuclei barcoding using 10X Genomics Chromium Single Cell 3' Reagent Kits (v3 Chemistry). 5000 nuclei were subjected to GEM generation and barcoding using Chromium Controller, according to the manufacturer's protocol. For cDNA amplification, 14 PCR cycles were used. Indexed cDNA libraries were pooled into a single pool and sequenced 4 times in Illumina NextSeq 550 to achieve >50,000 reads/nuclei.