GSM7992310: EC, Day 3, RNA-Seq, Replicate 1; Mus musculus; RNA-Seq
Cardiomyocytes were enriched by performing repeated centrifugation steps (20 x g, 2 minutes). The supernatant of the centrifugation step was used to isolate hematopoietic cells, endothelial cells, and fibroblasts. Hematopoietic and endothelial cells were isolated by magnetic separation. Cells not bound to the magnetic beads or columns were used for cardiac fibroblast enrichment by a plating step with uncoated dishes. After 40 minutes, predominantly DDR2-positive fibroblasts adhered and were used for RNA isolation. Cells extracted from three mice were pooled to generate enough cells for one of four biological replicate sequencing samples for each timepoint. Total RNA was isolated from CM, EC, HC and FB using the Qiagen (Hilden, Germany) miRNeasy Kit involving column DNAse digestion. RNA quantity was fluorometrically measured using the Qubit 4 (Thermo Fisher) RNA HS Assay Kit and integrity of the RNA was verified using the Agilent Bioanalyzer RNA Pico chips. Long RNA was converted into Illumina compatible libraries using the CATS Total RNA-seq Kit v2 with rRNA depletion (Diagenode). After the depletion, this procedure involves the fragmentation of RNA, 3 ́-polyadenylation and cDNA synthesis primed by oligo-T. Addition of a template switch oligo allows in combination with a suitable reverse transcriptase allows then the generation of double stranded cDNA with adaptor sequences for further amplification.