title: GSM9258342: AIYT-312_ChIP-Seq_HA-UzcR_Caulobacter_crescentus_NA1000∆uzcR;xylX::Pxyl_HA-uzcR; 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), the cell resuspensions 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 seconds to shear DNA fragments to an average length of 0.3–0.5 kbp and cleared by centrifugation at 14,000 rpm for 2 minutes 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-A agarose (Roche, www.roche.com) and 100 μg BSA. Five percent of the pre-cleared lysates were kept as total input samples (negative control samples). The rest of the pre-cleared lysates was then incubated overnight at 4°C with monoclonal rabbit Anti-HA Tag antibodies (Millipore, clone 114-2C-7) at a 1:400 dilution. Immuno-complexes were captured by incubation with Protein-A agarose beads (pre-saturated with BSA) during a 2 hour period at 4°C and then, washed 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). Immuno-complexes were then eluted from the Protein-A beads with two times 250 μL elution buffer (SDS 1%, 0.1 M NaHCO3, freshly prepared) and then, just like the total input samples, 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 30 μL of DNAse/RNAse free water. The immunoprecipitated chromatin was used to prepare sample libraries used for deep sequencing by Fasteris SA (Geneva, Switzerland). ChIP-Seq libraries were prepared using the DNA Sample Prep Kit (Illumina) following manufacturer instructions. Single-end run was performed on an Illumina Next-Generation DNA sequencing instruments (NextSeq High), 50 cycles (for HA-uzcR) and 75 cycles (HA-chvI), respectively, were performed and yielded several million reads per sequenced sample.