title: GSM4290266: RpoABC_ChIP-Seq_WT(MB1)_(AIYT-161); Caulobacter vibrioides NA1000; ChIP-Seq
library_construction_protocol: After resuspension of the cells in TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl) containing 10 mM of DTT, they were incubated in the presence of Ready-Lyse lysozyme solution (Epicentre, Madison, WI) for 10 minutes at 37°C, according to the manufacturer's instructions. Lysates were sonicated (Bioruptor® Pico) at 4°C using 15 bursts of 30 sec to shear DNA fragments to an average length of 0.3-0.5 kbp and cleared by centrifugation at 14,000 rpm for 2 min at 4°C. The volume of the lysates was then adjusted (relative to the protein concentration) to 1 ml using ChIP buffer (0.01% SDS, 1.1% Triton X-84 100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl) containing protease inhibitors (Roche) and pre-cleared with 80 μl of protein-G agarose (Roche, www.roche.com) and 100 μg BSA. The rest of the pre-cleared lysates was incubated overnight at 4°C with E. coli RNA polymerase antibody sampler kit (Cat 699907, Biolegend https://www.biolegend.com/fr-ch/products/e--coli-rna-polymerase-antibody-sampler-kit-15078) with the different antibodies mixed with a ratio of 1:1:1 (1:500 dilution). The immuno-complexes were captured after incubation with Protein-G agarose (pre-saturated with BSA) during a 2 h incubation at 4°C and they were washed subsequently with low salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), with high salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), with LiCl washing buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and finally twice with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA). The complexes were eluted from the Protein-G agarose beads with two times 250 μl elution buffer (SDS 1%, 0.1 M NaHCO3, freshly prepared) and then, incubated overnight with 300 mM NaCl at 65°C to reverse the crosslinks. The samples were then treated with 2 μg of Proteinase K for 2 h at 45°C in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5). DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1), ethanol-precipitated using 20 μg of glycogen as a carrier and resuspended in 50 μl of DNAse/RNAse free water.  Immunoprecipitated chromatin was used to prepare sample libraries used for deep-sequencing at Fasteris SA (Geneva, Switzerland). ChIP-Seq libraries were prepared using the DNA Sample Prep Kit (Illumina) following manufacturer instructions. TruSeq ChIP Library Preparation Kit
attributes.tag: GEO Accession
attributes.value: GSM4290266