GSM5285095: ChIP-Seq_ParB(WT)_Myxococcus_xanthus_SA4269(ParB_depletion_strain)_Sample1(AIYT-254); Myxococcus xanthus DK 1622; ChIP-Seq
After resuspension in TES buffer (10 mM Tris/HCl pH 7.5, 100 mM NaCl, 1 mM EDTA), the cells were incubated for 10 min at 37°C in the presence of Ready-Lyse lysozyme solution (Epicentre, Madison, WI) according to the manufacturer's instructions. The lysates were then sonicated (Bioruptor® Pico, Diagenode) at 4°C using 15 bursts of 30 sec to shear DNA fragments to an average length of 0.2-0.5 kbp and cleared by centrifugation for 2 min at 14,000 rpm and 4°C. The volume of the lysates was adjusted (relative to the protein concentration) to 1 ml using ChIP buffer (16.7 mM Tris/HCl pH 8.1, 167 mM NaCl, 0.01% SDS, 1.1% Triton X-84 100, 1.2 mM EDTA) containing protease inhibitors (Roche) and pre-cleared with 80 μl of Protein-A agarose (Roche) and 100 μg BSA. 2% of each pre-cleared lysate were reserved as total input (negative control) samples. The pre-cleared lysates were then incubated overnight at 4°C with monoclonal rabbit Anti-HA Tag anti¬bodies (Millipore, clone 114-2C-7) at a 1:400 dilutions. Immunocomplexes were captured by incubation with Protein-A agarose beads (pre-saturated with BSA) for 4 h at 4°C. After collection by centrifugation, the beads were washed with low-salt washing buffer (20 mM Tris/HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), with high-salt washing buffer (20 mM Tris/HCl pH 8.1, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), with LiCl washing buffer (10 mM Tris/HCl pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholate,) and finally twice with TE buffer (10 mM Tris/HCl pH 8.1, 1 mM EDTA). The immunocomplexes were eluted from the Protein-A agarose beads with two times 250 μL elution buffer (0.1 M NaHCO3, 1% SDS; freshly prepared) and, in parallel to 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 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 the manufacturer's instructions. Single-end runs with 50 cycles were performed on an NextSeq High (Illumina) next-generation DNA sequencing instrument.