title: NCCIT-RA-RNAP
design: NCCIT cells were collected and resuspended in digestion buffer (50 mM Tris-Cl, pH 7.6; 1 mM CaCl2, 0.2% Triton X-100, 5 mM butyrate, 1X protease inhibitor cocktail, 0.5 mM PMSF), followed by incubation with 0.3 U Micrococcal nuclease (MNase; Sigma-Aldrich) at 37°C for 5 min. The reaction was stopped with 50 mM EDTA and treated with RIPA buffer for 16 h, followed by incubation with an appropriate antibody (see below) and Dynabeads® Protein A beads (Invitrogen) for 16 h at 4°C. Chromatin-antibody-Dynabeads® Protein A complexes were washed with RIPA buffer supplemented with 0.3 M NaCl, lithium chloride (LiCl) buffer and TE buffer, consecutively, and were incubated with proteinase K at 65°C for 16 h. DNA from the complexes was purified via phenol/chloroform extraction and concentrated by ethanol precipitation. The resulting DNA pellets were dissolved in TE buffer for further analyses. Immunoprecipitated DNA was used to construct paired-end ChIP-sequencing libraries using the NEBNext® UltraTM DNA Library Prep Kit for Illumina®. ChIP reactions using rabbit IgG, and from untreated and RA-treated NCCIT were used as normalization controls. Two biological replicates were prepared from each condition.