GSM8464572: control_2; Mus musculus; RNA-Seq
Sample preparation and sequencing of murine cardiac interstitial cells was performed according to the detailed protocol published previously with only minor modifications[30]. In brief, rapidly after sacrificing the animals by cervical dislocation, the still beating heart was directly placed in ice-cold HBSS where atria and large vessels were dissected. After chopping the heart into small pieces, enzymatic digestion was initiated in two rounds of 15min duration at 37°C using collagenase type II ( Worthington Biochemical Corporation,  # LS004177). The single-cell suspension was subsequently passed through a 40 µm cell strainer, washed and red blood cell lysis performed. Dead cells were removed by Dead Cell Removal MicroBeads   (Miltenyi Biotec, 130-090-101) binding to MACS  LS columns (Miltenyi Biotec, 130-042-401). Prior to loading the 10x platform, live nucleated cells (DRAQ5+, propidium iodine-) were sorted using a FACSAriaTM IIu (BD) cell sorter. Following washing and resuspending in PBS, cells were counted manually using trypan blue and a Neubauer chamber. We aimed to load about 5,000 cells per lane on a Chromium Next GEM Chip (10x Genomics, 1000127), that was placed into a 10X Chromium Controller (10X Genomics). The cDNA output was amplified and library construction was performed according to the manufacturer's instructions using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, 1000269) and Dual Index Kit TT Set A (10x Genomics, 1000215). Respective library quantification and quality controls were performed using an Agilent 2100 Bioanalyzer and in addition a Qubit HS Assay. Indexed libraries were equimolarly pooled resulting in two sequencing runs (control1+HFpEF1; control2+HFpEF2) using a High Output kit v2.5 (Illumina, 20024907) and a NextSeq® 550 (Illumina) sequencer.