GSM6876590: ZM532, RM, Zur, rep1; Zymomonas mobilis; ChIP-Seq
Formaldehyde was added to cultures to a final concentration of 1% and cells were crosslinked for 25 min at 30 °C with 100 rpm rotation. Quench formaldehyde was carried out by adding 0.25 M glycine. After crosslink, cells were collected by centrifugation at 4,000 rpm for 10 min and washed twice with 1× ice-cold Phosphate Buffered Saline. The crosslinked pellets were stored at -80 °C until use, or resuspended in 1 ml FA lysis buffer (50 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) per 50 ml cultures containing 2 mg ml-1 lysozyme and 1× Protease Inhibitor Cocktail II (Abcam), and then incubated at 37 °C for 30 min to lyse cells. Sonication was used to shred genomic DNA into 200-500 bp fragments, which were then centrifuged at 14,000 rpm for 15 min. The supernatant was retained and either used for immunoprecipitation or stored at -80 °C. Before starting the IP assays, ~ 2% of the supernatant was kept as the input sample, and the remaining extract was pre-cleared with 30 μl agarose protein G beads (CST, 9007s) at 4 °C for 1 h to reduce nonspecific background. The beads can be pelleted by centrifuging at 6,000 rpm for 1 min as per manufacturer's instructions. 4 μg ml-1 anti-Zur or 10 μg ml-1 anti-Fur monoclonal antibody was added to the supernatant above and incubated at 4 °C overnight. After that, 30 μl agarose protein G beads were added and rocked at 4 °C for 1 h. After centrifugation, the beads were washed twice with 1 ml FA lysis buffer, once with high salt buffer (lysis buffer + 500 mM NaCl), once with LiCl buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 250 mM LiCl, 0.5 % Na deoxycholate), and twice with TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA). Finally, the beads were incubated with 100 μl elution buffer (50 mM Tris, pH 7.5, 10 mM EDTA, 1% SDS) for 20 min at 65 °C and washed once with 150 μl of TE + 0.67% SDS. The supernatant of eluted DNA complexes above was collected and then blended. In addition, 150 μl of TE + 0.67% SDS was added to the input sample. To reverse the crosslinking, all IP and input samples were incubated at 65 °C overnight. After treated with 1 μl of RNase A (10 mg ml-1) and 5 μl of Proteinase K (10 mg ml-1) at 37 °C for 1 h respectively, the immunoprecipitated chromatin was purified using phenol-chloroform-isoamyl alcohol method and resuspend in 50 μl TE buffer pH 7.5. The DNA-sequencing library was generated by NEBNext Ultra II DNA Library Prep Kit (NEB) as per manufacturer's instructions and subsequently sequenced on the Illumina HiSeq X Ten platform in 150PE mode.