title: GSM5538290: Mock DNA; Escherichia coli; ChIP-Seq
library_construction_protocol: For the Mock-DNA fragment size analysis, a 100 µl aliquot of the clarified lysate was first treated with proteinase K (Thermo-Fisher Scientific) at 0.125 mg/ml in the presence of 1% SDS for 4 h at 37°C followed by DNA de-crosslinking by incubation at 65°C for 6 h. The Mock-DNA material was purified using Qiagen PCR purification kit (Qiagen) and quantified by Nano-Drop spectrophotometer. The DNA fragment size distribution was assessed by DNA electrophoresis. For the preparation of RNAP-ChIP DNA, the remaining lysate (~900 µl) was purified by immunoprecipitation (IP). The lysate was mixed with 10 µl of IgG-agarose (GE Healthcare) and incubated at 4°C overnight on a rotating mixer in the presence of Protease Inhibitor Cocktail (Sigma). Then affinity resin was washed consecutively with 1 ml each of the following solutions: 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 2 M NaCl; 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 1 M NaCl; 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 200 mM NaCl; and twice with RIPA buffer (50 mM Tris-HCl pH 7.4, 140 mM NaCl, 1% NP-40, 0.1% Deoxycholate, 1% SDS). All wash procedures were carried out by resuspending the beads in the wash solution and inverting the tube several times, followed by brief centrifugation and removing the supernatant by vacuum aspiration. The last wash was done for 20 min at 4°C on a rotary mixer. The RNAP-DNA crosslinks were eluted by incubation with ChIP Elution Buffer (10 mM Tris pH 8.0, 30 mM EDTA, 0.1% SDS) at 65°C for 4 hours (or O/N) on a shaker at 800-900 rpm. The resulting material was treated with Proteinase K (Thermo Fisher Scientific) (0.2 mg/ml) at 37°C for 3-5 h, followed by IP-DNA decrosslinking by incubation at 95°C for 2h. The IP-DNA was finally purified using ChIP DNA Cleaning & Concentrator kit (Zymo Research). A 50-100 ng of IP-DNA or Mock-DNA were end repaired by a mix of T4 DNA polymerase (NEB), T4 PNK (NEB), and Klenow DNA polymerase (NEB) and purified by Qiaquick PCR DNA purification kit (Qiagen). The eluted DNA material was A-tailed by Klenow Fragment (3ʼ to 5ʼ exo minus) (NEB) followed by purified by MinElute PCR purification Kit (Qiagen). Illumina Multiplex Adapters (MPA) were ligated with Quick DNA ligase (NEB) and DNA was purified by AMPure XP beads (Beckman Coulter). Resulting library was separated by agarose electrophoresis with following size selection of DNA bands corresponding to 220 bp, which were excised by Gel X-tracta tool (USA scientific) and purified with Gel Extraction Kit (Qiagen). The purified DNA material was PCR-amplified 18 cycles using Phusion polymerase (NEB) and purified by MinElute PCR purification kit (Qiagen). DNA sequencing was performed by Illumina HiSeq 50+50 bp paired-end protocol.