DNase I Hypersensitivity Mapping in TGFβ-Treated Cells

DNase I hypersensitivity mapping was performed according to ref. ^{98 (link)} with brief modifications. NMuMG cells were either vehicle or TGFβ-treated for 2 h. The cells were trypsinized and pelleted before washing and resuspension in buffer A (15 mM Tris-Cl (pH 8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 0.5 mM spermidine, 0.15 mM spermine). Nuclei were extracted by adding buffer A containing NP-40. The nuclei were washed with buffer A and resuspended in pre-warmed lysis buffer at a concentration of 5 × 10⁶/ml and then digested with 75 units of DNase I (Roche) for 5 min at 37 °C. The reactions were terminated by the addition of an equal volume of stop buffer and incubated at 55 °C. After 15 min, proteinase K (final concentration of 20 μg/ml) was added to each digestion reaction and incubated for 16 h at 55 °C. DNA was extracted by careful phenol-chloroform purification using phase-lock gel. DNA fragments of 50–300 bp were selected using low-melting agarose gel and was purified using GenElute Gel Extraction Kit (Sigma-Aldrich). DNA was again purified using MinElute PCR Purification kit and samples were sequenced using Illumina NextSeq 500 system (Illumina) with 75 bp paired-end reads at the Genomic Unit of CABIMER (Sevilla, Spain).