title: ChIP-seq identification of CrsL (MSMEG_5890) binding sites in Mycobacterium smegmatis
design: ChIP-seq identification of CrsL (MSMEG_5890) binding sites in Mycobacterium smegmatis
library_construction_protocol: 100 ml of bacterial culture was crosslinked with 1% formaldehyde (final conc.), formaldehyde was added directly to the medium) for 30 minutes at 37°C in the shaker. Formaldehyde was quenched by glycine (0.125 M final conc.) added for 5 minutes at 37°C in the shaker. Bacteria were centrifuged for 5 min at 9000 rpm, 4°C, pellet washed with 10 ml of 1x PBS, bacteria centrifuged again and pellet immediately frozen at -80°C. M. smegmatis mc2 155 strain LK3051 was grown at 37°C in Middlebrook 7H9 medium with 0.2% glycerol and 0.05% Tween 80. Cells were inoculated to OD600 0.1 in 100 ml of 7H9/Tween/glycerol, anhydrotetracycline was added after 3 hours of growth (10 ng/ml final concentration). After additional 3 hours (totals 6 hours of growth) cells were harvested. Bacterial pellet was resuspended in 3 ml of ice cold RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% dexycholate, 0.1% SDS, 50 mM Tris-Cl pH 8.0, 5 mM EDTA) with protease inhibitors cocktail (Sigma-Aldrich, 5 ul/1 ml) and 0.1 M PMSF (5 ul/1 ml) and sonicated 18x10 seconds with 1 min pauses on ice between cycles. Lysate was centrifuged 9000 rpm at 4°C for 15 min and pellet discarded. 2 mg of supernatant was incubated with 20 ul of M2 anti-FLAG resin (Sigma-Aldrich) overnight at 4°C. M2 resin was rinsed twice with RIPA, four times with 100 mM Tris-HCl, pH 8.5, 500 mM LiCL, 1% Triton X-100, 1% deoxycholic acid, twice again with RIPA and twice with TE. Protein-DNA complexes were eluted with 1% SDS for 10 minutes at 65°C, decrosslinked in the presence of 200 mM NaCl for 5 h at 65°C, treated with 100 ug/ml RNAse A for 1 hour at 37°C and 20 µg proteinase K for 30 min at 45°C. DNA was purified with Qiagen PCR purification kit and eluted into 100 ul EB. 40 ul of immunoprecipitated DNA sample or 10 ng of DNA input were used for library construction according to the NEXTflex™ ChIP-Seq Kit manual including the Size-Selection Cleanup step B2.