1. Monoclonal antibodies specific for HA from Sigma Aldrich and the catalog number is 89658 2. Monoclonal antibodies specific for RNAP beta subunit from Neoclone, Madison, WI. The antibody clone was 8RB13 and the catalog number is WP023 3. Monoclonal antibodies specific for RNAP sigma subunit from Neoclone, Madison, WI. The antibody clone was 2G10 and the catalog number is W0004 The cell pellet was resuspended in TE with protease inhibitors. Cells were lysed and DNA was sheered to 100bp fragments in the Covaris focused ultrasonicator. Cell lysates (in 50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protein-nucleic acid complexes were incubated with immobilized HA specific antibodies to immunoprecipitate CarD containing complexes from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA from the Mc2155 attB::pmsg431. RNAPb and RNAPs containing complexes were immunoprecipitated  from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain using specific monoclonal antibodies immobilized on Gammabind sepharose. Co-precipitated  complexes were washed 4 times in 10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA with increasing salt. Co-precipitated complexes were eluted in 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% sodium dodecyl sulfate at 65 oC. Eluate crosslinks were reversed by incubating overnight at 65 oC. The samples were treated with 100 ug/ml of proteinase K to each sample and incubate at 37 oC for 2 hours. DNA was extracted by phenol:chloroform:IAA (25:24:1) extraction, precipitated with ethanol, and resuspended in water.
GEO Accession
GSM1171540