GSM1936197: ssrS_1_IP; Escherichia coli str. K-12 substr. MG1655; ChIP-Seq
Cross-linked cells were harvested by centrifugation, washed thrice with ice-cold TBS (pH 7.5), resuspended in 1 ml lysis buffer [10 mM Tris (pH 8.0), 20% sucrose, 50 mM NaCl, 10 mM EDTA, 20 mg/ml lysozyme and 0.1 mg/ml RNase A] and incubated at 37°C for 30 min. After lysis, 3 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% sodium dodecyl sulphate (SDS) and PMSF (final 1 mM)] was added and the DNA sheared to an average size of ~250 bp using a Bioruptor (Diagenode). Insoluble cellular matter was removed by centrifugation for 10 min at 4°C. An 800 μl aliquot was incubated with 20 μl Protein A/G UltraLink Resin (Pierce) on a rotary shaker for 45 minutes at room temperature. The supernatant was removed and incubated with mouse monoclonal antibody (Neoclone cat. no. WP009) and 30 μl Protein A/G UltraLink Resin (pre-incubated with 1mg/ml BSA in TBS), on a rotary shaker at room temperature for 90 min. Samples were washed once with IP buffer, twice with IP buffer + 500 mM NaCl, once with wash buffer [10 mM Tris (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet P-40 and 0.5% sodium deoxycholate] and once with TE (pH 7.5). Immunoprecipitated complexes were eluted in 100 μl elution buffer [10 mM Tris (pH 7.5), 10 mM EDTA and 1% SDS] at 65°C for 20 min. Immunoprecipitated samples and the sheared DNA from the Bioruptor were uncrosslinked in elution buffer containing 0.8 mg/ml Pronase at 42°C for 2 h followed by 65°C for 6 h. DNA was purified using the phenol-chloroform method. Libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM1936197