GSM7992212: EC, Day 1, smallRNA-Seq, Replicate 3; Mus musculus; ncRNA-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. Small RNAs were converted into Illumina compatible libraries using the NEB Next small RNA library Kit from New England Biolabs (NEB), which involves 3´-ligation and 5´-ligation to small RNAs and therefore enriches for 5´-monophosphorylated and 3 ́-hydroxylated small RNAs, such as miRNA. Due to the varying RNA amounts available for each cell type, we used the following parameters for input of total RNA and PCR cycles: CM-500ng-14cycles, EC-50ng-16cycles, HC-150ng-15cycles, FB-150ng-15-cycles. After library amplification, we cut each library from 10% TBE (Tris-Borate-EDTA) PAGE gels, extracting the library size of approx. 140-150bp which corresponds to 20-30nt miRNAs. Libraries were then quantified using the Qubit dsDNA HS Assay (Thermo Fisher).