GSM9150853: LV tissue, C57BL/6N mouse, HFpEF diet, Biological Replicate 2; Mus musculus; RNA-Seq
Left ventricle tissue samples were homogenized using a TissueLyzer II (Qiagen) in homogenization buffer (250 mmol/L sucrose, 25 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L Tris-HCl, 1 mmol/L dithiothreitol, and 1× protease inhibitor). The lysate was filtered through a 40-µm strainer (Greiner) and centrifuged at 1200g for 5 minutes at 4 °C. The supernatant, containing cytoplasmic total RNA, was collected and stored at −80 °C. The remaining nuclei pellet was resuspended in storage buffer (1× PBS, 4% BSA, 0.2 U/µL Protector RNase Inhibitor). Nuclei were stained with NucBlue Live ReadyProbes Reagent (Thermo Fisher Scientific), and Hoechst-positive nuclei were purified by fluorescence-activated cell sorting (Aria, BD Biosciences). Fluorescence-activated cell sorting–sorted nuclei were processed on the Chromium iX (10× Genomics) with a targeted recovery of 10 000 nuclei per run, following the manufacturer's protocol. The 3′ gene expression libraries were prepared using the Chromium Single-Cell Reagent Kits v3.1 (10× Genomics). Libraries were sequenced on a NovaSeq 6000 (Illumina) at a depth of 30 000 to 40 000 read pairs per nucleus.