GSM5775729: KO RNA-seq rep2; Mus musculus; RNA-Seq
Cardiac fibroblasts isolated from WT and Acta2fl/fl mice were transduced with Adeno-Cre, treated with TGFβ for 2 days, and then subjected to RNA isolation using a Direct-zol RNA Microprep Kit (Zymo). cDNA libraries were constructed using the NEBNext single cell/low input RNA library prep kit for Illumina (E6420) from New England BioLabs. The cDNA libraries were sequenced on the Illumina Hi-seq platform using 150 bp paired-end sequencing. RNA-seq reads that passed filters were trimmed to remove low-quality reads and adaptors by TrimGalore-0.6. The quality of reads after filtering was assessed by fastQC, followed by alignment to the mouse genome (MM10) by STAR (2.5.3a) with default parameters. Individual mapped reads were adjusted to provide TPM (transcripts per million) values with mouse genome as the reference. Heart tissue samples were minced and digested in DMEM containing 0.75U/ml Collagenase D (Roche 11088866001), 1.0U/ml Dispase II (Roche 10165859001), and 1 mM CaCl2 at 37oC for 40 min. The slurry was then passed through a 100-μm cell strainer and then a 40-μm cell strainer. Cells were collected by centrifugation at 350 × g for 10 min. The cell pellet was then resuspended in PBS. Tcf21 lineage-traced eGFP+ cardiac fibroblasts were sorted on FACSaria II (BD Biosciences). Gates were made based on WT eGFP- control. Sorted eGFP+ cardiac fibroblasts were used for RNA-seq and ATAC-seq analyses