GSM5514674: DRIP-Seq, EcTopoI 14-kDa CTD overexpression and Rif 100 mkg/ml for 20 min, replicate 3; Escherichia coli; OTHER
Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. To shear nucleic acids, sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). As a control, an aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37oC and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. DNA sequencing was performed using Illumina NextSeq with 75+75 bp paired-end protocol. Libraries preparation and sequencing were performed at Skoltech Sequencing Core Facilities. DRIP-Seq was performed in triplicate.