GSM1310816: delta-phoP mutant   [ChIP-Seq]; Mycobacterium tuberculosis H37Rv; ChIP-Seq
Cross-linking was quenched by addition of glycine (125 mM). Cells were then washed twice with Tris-buffered saline (TBS, 20 mM Tris-HCl pH 7.5, 150 mM NaCl), resuspended in 4 ml immunoprecipitation (IP) buffer (50 mM Hepes-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail from Roche) and sonicated to shear DNA using Bioruptor (Diagenode). Cell debris was removed by centrifugation and the supernatant used in IP experiments. Nucleo-protein extracts were incubated with 50 μl of rabbit polyclonal anti-PhoP antibodies at 4°C for 2 days on a rotating wheel. Complexes were subsequently precipitated with Dynabeads (Dynal, anti-rabbit, Invitrogen) for three hours at 4°C. Beads were washed twice with IP buffer, once with IP buffer plus 500 mM NaCl, once with buffer III (10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% sodium deoxycholate), once with Tris-EDTA buffer pH 7.5. Elution was performed in 50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS for 40 minutes at 65°C. Samples were finally treated with RNAse A for one hour at 37°C and cross-links were reversed by incubation for two hours at 50°C and for eight hours at 65°C in 0.5x elution buffer with 50 μg Proteinase K (Eurogentec). DNA was purified by phenol-chloroform extraction and quantified by Nanodrop and Qubit fluorometer according to the manufacturer’s recommendations (Invitrogen). ChIP-Seq Sample Preparation Kit (Illumina), according to the protocol provided by the manufacturer
GEO Accession
GSM1310816