GSM5904363: HVO_oxsRHA_1_IP; Haloferax volcanii; ChIP-Seq
ChIP-seq samples were prepared as the previous method with modifications (Wilbanks et al., 2012; DOI: 10.1093/nar/gks063). Briefly, to crosslink, 37% formaldehyde was added to the culture at the final concentration of 1% and the cell culture was incubated on a rocking platform for 20 min at room temperature. A final concentration of 0.125 M glycine was added to stop the crosslinking reaction and the whole cells were washed three times with cold basal salts buffer followed by storage at -80°C until sonication. The cell pellet was thawed and resuspended in 800 μl lysis buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, pH 7.5) containing protease inhibitor cocktail (Thermo Scientific) to shear DNA by Bioruptor 300 sonication system (Diagenode) with 15 cycles of 30 s on and 90 s off at high magnitude. The sheared DNA was monitored to confirm the genomic DNA was a smear between 200-800 bp with the highest concentration of fragments ~500 bp by 1.2% (w/v) agarose gel electrophoresis. For immunoprecipitation, the sheared DNA was immediately incubated overnight with the complex anti-HA tag antibody (Abcam, ab9110) and Dynabeads protein A (Invitrogen) at 4°C. Enriched DNA/OxsR complexes were eluted by adding 50 µl elution buffer (50 mM Tris, 10 mM EDTA, 1% SDS (w/v), pH 8.0) and incubation at 65°C for 10 min. Reverse crosslinking was performed by incubating in TE/SDS (10 mM Tris, 1 mM EDTA, 1% SDS) overnight at 65°C. DNA, of which the RNA was removed, was subsequently extracted by a phenol-chloroform method. Library preparation and deep sequencing were carried out at Duke Sequencing and Genomics Technologies core facility.