GSM8263032: Col1a1 GFP+ Cardiac Fibroblasts 3 days after MI Replicate 2; Mus musculus; RNA-Seq
Mouse cardiac interstitial cells (CIC) from infarcted (1 to 6 days post-infarction) 8-10 week old mice heart 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. For Col1a1-GFP+ reporter mouse, GFP+/CD31-/CD45- cardiac fibroblasts (CF) were FACS-sorted in 1X PBS, 0.05% BSA. Bulk RNA-seq was performed following a massively parallel RNA single-cell sequencing (MARS-seq) protocol adapted for bulk RNA-seqwith minor modifications. Briefly, poly-A RNA was extracted with Dynabeads Oligo (dT) (Ambion) and reverse-transcribed with AffinityScript Multiple Temperature Reverse Transcriptase (RT) (Agilent) using poly-dT oligo primers (IDT) carrying a 7 bp index. Samples were subjected to linear amplification via IVT using the HiScribe T7 High Yield RNA Synthesis Kit from 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 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. Sequencing was carried out in an Illumina NextSeq 500 using paired-end, dual-index sequencing (Read1: 68 cycles; Read2: 15 cycles; i7 index: 8 cycles) at a depth of 10 million reads per sample.