GSM5775726: KO ATAC-seq rep1; Mus musculus; ATAC-seq
ATACseq was performed as previously described. Briefly, cardiac fibroblasts isolated from WT and Acta2fl/fl mice were transduced with Adeno-Cre, treated with TGFβ for 2 days, and then 10,000 cells were lysed on ice for 20 minutes to isolate the nuclei. Isolated nuclei were then incubated with the Tn5 transposase (TDE1, Illumina) and tagmentation buffer at 37°C for 30 minutes with shaking on a thermomixer at 700 rpm. Tagmentated DNA was purified using MinElute Reaction Cleanup Kit (Qiagen). PCR was performed to amplify the ATACseq libraries using Illumina TrueSeq primers and multiplex by indexes primers. After the PCR reaction, libraries were purified with the 1.1X AMPure beads (Beckman). The ATACseq libraries were sequenced on the Illumina Hi-seq platform using 150 bp paired-end sequencing. Sequencing reads of all samples underwent adapter removal using TrimGalore-0.6, followed by quality assessment using FastQC. Reads were then aligned to the mouse reference genome MM10 using Bowtie 2.3 with the following options: – very-sensitive -X 2000 – no-mixed – no-discordant. Only unique alignments within each sample were retained in subsequent analysis. Moreover, alignments resulting from PCR duplicates or located in mitochondria were excluded. The mouse genome was tiled with consecutive non-overlapping 300bp bins. The accessibility of each of the 300bp bins was assessed by the number of fragments per million mapped (FPM) that was aligned to the bin. ATAC-seq peaks were called separately for each sample by MACS2 (48) with the following options: – keep-dup all – nolambda – nomodel. The identification of transcriptional factor motifs in peaks was evaluated using HOMER (http://homer.ucsd.edu/homer/motif/). 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