GSM4760641: Healthy Right Ventricle 3; Homo sapiens; RNA-Seq
Mouse cardiac interstitial cells (CIC) were obtained by enzymatic digestion. Erythrocytes were removed using RBC lysis buffer. A positive selection of CIC, enriched in Cardiac fibroblasts, was performed using LS columns and Feeder Removal MicroBeads (Miltenyi) according to the manufacturer's instructions. Bulk RNA-seq was performed following MARS-seq adapted for bulk RNA-seq with minor modifications. Briefly, poly-A RNA was extracted with Dynabeads Oligo dT (Ambion) and reverse-transcribed with AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) using poly-dT oligos (IDT) carrying a 7-bp index. Up to 8 samples with similar overall RNA content were pooled together and subjected to linear amplification via IVT using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs (NEB)). The resulting antisense RNA was fragmented into 250-350 bps fragments with RNA Fragmentation Reagents (Ambion) and dephosphorylated with 1U FastAP (Thermo Scientific) for 15 min at 37 °C. Partial Illumina adaptor sequences were ligated with T4 RNA Ligase 1 (NEB) followed by a second reverse transcription. Full Illumina adaptor sequences and a secondary barcode were added during library amplification with KAPA HiFi DNA Polymerase (Kapa Biosystems). Libraries were quantified using a Qubit 3.0 Fluorometer and their size profiles examined in Agilent's 4200 TapeStation System. For Col1a1-GFP+ reporter mouse GFP+/CD31-/CD45- cardiac fibroblasts (CF), GFP-/CD31+/CD45- endothelial cells and GFP-/CD31-/CD45+ bone marrow-derived cells were FACS-sorted in Lysis/Binding Buffer (Ambion).
GEO Accession
GSM4760641