title: GSM2723766: Exp 2_IP sample; Escherichia coli str. K-12 substr. MG1655; ChIP-Seq
library_construction_protocol: For immunoprecipitation, 800 μl of collected chromatin (~1000 μg of protein) were incubated on a rotating wheel over night at 4°C with 10 μg of either rabbit anti-Dps antibodies (experimental sample) or rabbit pre-immune IgG (negative control). Next day 30 μl of Ultra Link Protein A/G beads (Thermo Scientific, USA) were added to the samples, and incubation was allowed for a further 2 hours at 4°C on a rotating wheel. The beads were washed 3 times with 1 ml of the low-salt buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton X-100, 1xPIC) and then with 1ml of the high-salt buffer (500 mM NaCl). Immunoprecipitated DNA-protein complexes were removed from the beads by 3 h shaking at 65°C and 1000 rpm in 110 μl of freshly prepared elution buffer, containing 100 mM NaHCO3 and 1% SDS. After centrifugation at 3000 rpm for 5 minutes, 100 μl of the supernatant was transferred to a new tube, and the DNA was purified with a PCR Purification Kit (Qiagen, Germany). The DNA concentration was measured on Qubit 2.0 using Qubit dsDNA HS Assay kit. A total of 8-10 identical samples were combined and concentrated to a volume of 30-50 μl containing 5-10 ng of the DNA. ChIP-seq libraries were prepared from 5-10 ng of the DNA samples with the NebNext® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) following the manufacturer’s instructions. For the final amplification of the library 15 PCR cycles were used. Size distribution and concentration of the amplicons was checked on the Bioanalyzer 2100 (Agilent, USA). In the first experiment, the maximum was at about 300 bp, and ChIP libraries were sequenced using 50 nt single-end read protocol on the Illumina HiSeq system (Illumina, USA) of the Genomics Facility in the Centre for Genomic Regulation (Barcelona). In the second experiment, the maximum was at about 450 bp, and samples were sequenced using standard paired-end 2*150 nt protocol on the MiSeq system (Illumina, USA) in the Immanuel Kant Baltic Federal University (Kaliningrad).
attributes.tag: GEO Accession
attributes.value: GSM2723766