GSM7036802: Donor 3, Septum, snRNA-seq; Homo sapiens; RNA-Seq
Single nuclei isolation was performed on the heart tissue aliquots as described in Litvinukova et al. 2020 (PMID:  32971526), with some modifications. Single heart aliquots were kept on dry ice until being transferred into a precooled 2 mL dounce homogenizer (Sigma) with 2 mL homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1x protease inhibitor, 0.4 U/µl, RNaseIn, 0.2 U/µl SUPERaseIn, 0.1% Triton X-100 in nuclease-free water). Samples were dounced 25 times with pestle A (loose) and 15 times with pestle B (tight), filtered through a 40-µm cell strainer, and centrifuged (500g, 5 minutes, 4ºC). Supernatant was discarded and the nuclei pellet was suspended in nuclei resuspension buffer (1x PBS, 1% BSA, 0.2 U/µL RnaseIn) and stained with NucBlue Live ReadyProbes Reagents (ThermoFisher). Hoechst-positive nuclei were enriched using fluorescence-activated cell sorting (FACS) on the FACSAria (BD Biosciences), obtaining between 172,500 and 350,000 nuclei while targeting a maximum of 350,000. Nuclei were sorted into 0.75 ml of resuspension buffer. Flow-sorted nuclei were counted in a C-Chip Disposable Hemocytometer, Neubauer Improved (INCYTO) before commencing with library preparation. A portion of the sorted nuclei suspension was removed and brought to a concentration of between 700 and 1,200 nuclei per microliter. An appropriate number of nuclei were loaded on the Chromium controller (10X genomics) in order to target between 6,000-8,000 nuclei, according to V3 of the manufacturer's instructions for the Chromium Next GEM Single Cell 3ʹ Reagent Kits (10X Genomics). 3' gene expression libraries were amplified with 15 cycles during sample index PCR. QC was performed on 3' gene expression cDNA and final libraries using a Qubit Fluorometer (ThermoFisher) and an Agilent 2100 Bioanalyzer (Agilent). single nucleus RNA-seq library,  3' mRNA libraries (10x Genomics)