Chip dna clean and concentration kit

ChIP assay was performed using ChIP-IT Express Kit (Active Motif) according to the manufacturer’s instructions with minor modifications. Briefly, the fixed cells were lysed and chromatin was sheared using an enzymatic shearing cocktail for 10 min at 37 °C. The sheared chromatin was mixed with 3 μg of antibody and 40 μL of magnetic beads (Dynabeads M-280 sheep anti-mouse IgG or Dynabeads ProteinG (Invitrogen)) and incubated with rotation at 4 °C overnight. After IP, DNA was recovered by incubation with elution buffer (10% SDS, 300 mM NaCl, 10 mM Tris–HCl, and 5 mM EDTA, pH 8.0) at 65 °C for 6 h. Recovered DNA was purified using ChIP DNA Clean and Concentration Kit (Zymo Research) and was subjected to ChIP-quantitative PCR (qPCR).

ChIP was performed as previously described (75 (link)) with slight modifications. Briefly, chromatin was cross-linked with 1% formaldehyde, cells were incubated in lysis buffer (1% SDS, 0.01 M EDTA, 0.05 M Tris pH 8.1, 0.1 mM PMSF in isopropanol) and DNA was fragmented into approximately 500-bp fragments using a Bioruptor (Diagenode Pico Bioruptor). Aliquots of lysates containing 13 μg chromatin were used for immunoprecipitation using anti-MRTFA (Santa Cruz Biotechnology, sc-21558) or goat IgG (76 (link)). Cross-links were reversed by heating at 65°C in the presence of 0.2 M NaCl and DNA fragments were recovered using the ChIP DNA Clean and Concentration Kit from Zymo Research (catalog D5205). Primers for the promoter region (Supplemental Table 2) were located within 400 bp upstream of the transcription start site (52 (link)).