GSM8173903: AS6_rep1; Homo sapiens; OTHER
Myocardial tissue was obtained with a scalpel from the basal septum of the left ventricle from patients (N=11) undergoing surgical aortic valve replacement for AS or unmatched donor hearts from 2011-2012 (N=9; hearts not used for transplantation) and immediately snap frozen in liquid nitrogen. The tissue was subsequently stored in a -80°C freezer until being used for analysis. Briefly, frozen ventricular tissue was mounted onto a CryoStat using Optimal Cutting Temperature (OCT) compound, followed by 100 µm sectioning. OCT was removed, and sections were placed in cold lysis buffer, followed by gentle dounce homogenization to release intact nuclei. After pelleting at 40g for 1 minute to remove large debris, the suspension underwent sequential filtering using 100 µm and 20 µm filters. The filtered suspension was centrifuged at 550g for 5 minutes, washed in a nuclear wash buffer, and recentrifuged at 550g for 5 minutes. The nuclear wash buffer was removed via aspiration and the nuclei were resuspended in cold nuclear resuspension buffer containing RNase inhibitors. Nuclei were stained with trypan blue and counted using a hemocytometer. Nuclei were loaded into a 10X Genomics microfluidic platform (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression), targeting an estimated recovery of 8,000 nuclei per sample. Libraries were processed according to the manufacturer's instructions. The sequencing files were de-multiplexed using Cellranger-ARC mkfastq (v2.0.0). The RNA reads were then trimmed using Cutadapt (v2.8) with default parameters to remove homopolymers (A30, T30, G30, and C30) and the template switch oligo (CCCATGTACTCTGCGTTGATACCACTGCTT) and its complement (AAGCAGTGGTATCAACGCAGA GTACATGGG). To generate an RNA count matrix, Cellranger (v7.1.0) count was used with default settings to align the trimmed reads to the human genome (GRCh38) and CellBender (v2.0) was used with default settings to filter samples for ambient RNA byproducts and call valid cell barcodes. Single-nuclear RNA and ATAC seq (10X Multiome kit)