GSM2830260: 54_3; Native Cmr, basal cAMP levels; Mycobacterium tuberculosis variant bovis BCG str. Pasteur 1173P2; ChIP-Seq
A total of 50 ml culture volume of mid-log phase bacterial culture was cross-linked with 1% formaldehyde, for 30 min at room temperature, on a slow rocking platform. Cross-linking was quenched by addition of 250mM glycine and incubation for 15 min at room temperature with slow rocking. The cells were harvested, washed with cold PBS and resuspended in 0.5-0.6 ml of Buffer I (20 mM HEPES pH 7.9, 50 mM KCl, 0.5 M DTT, 10% glycerol) with protease inhibitor cocktail (Sigma). Bacterial cells were lysed and DNA sheared by sonication for 25 min (Covaris). The salt concentration of the cleared cell lysate was adjusted to a final concentration of 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40 (IPP150 buffer). Immunoprecipitation was carried out by incubation of lysate with 10ul of Cmr antiserum, on nutator at 4°C. A total of 50ul of Protein A agarose beads were rinsed with IPP150 buffer, and added to the lysate-antiserum mixture. Protein A agarose-lysate-antiserum mixture was incubated at 4°C for 30 min and room temperature for 1.5 h. The beads were washed at least five times with IPP150 buffer followed by two washes with TE buffer (10mMTris HCl pH 8.0, 1mM EDTA).DNA from agarose beads was eluted by incubation with 150ul elution buffer (50 mMTris HCl pH 8.0, 10mM EDTA, 1% SDS) at 65°C for 15 min. A second elution was carried out by incubation of pellet with 100ul of TE buffer+ 1% SDS at 65°C for 5 min. Both elutions were pooled and de-crosslinked by incubating with 1 mg/ml Proteinase K at 37°C for 2 h and 65°C overnight. DNA was purified using PCR purification kit (QIAGEN). Standard manufacturer (Illumina) recommended protocol was used for library construction
GEO Accession
GSM2830260