title: GSM3674392: PchR1; Pseudomonas aeruginosa PAO1; ChIP-Seq
library_construction_protocol: Wild-type P. aeruginosa containing empty pAK1900 or pAK1900-TFs-VSVG/FLAG was cultured in LB medium supplemented with ampicillin until mid-log phase (OD600 = 0.6), then treated with 1% formaldehyde for 10 min at 37℃. Cross-linking was stopped by the addition of 125 mM glycine. Bacterial pellets were washed twice with a Tris buffer (20 mM Tris– HCl [pH 7.5] and 150 mM NaCl), re-suspended in 500 μl IP buffer (50 mM HEPES–KOH [pH 7.5], 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and mini-protease inhibitor cocktail (Roche), and then subjected to sonication to produce 100–300 bp DNA fragments. Insoluble cellular debris was removed by centrifugation at 4℃ and the supernatant was used as the input sample in IP experiments. Both control and IP samples were washed with protein A beads (General Electric) and then incubated with 50 μl agarose-conjugated anti-VSV antibodies (Sigma) in IP buffer. Washing, crosslink reversal, and purification of the ChIP DNA were conducted as previously described. DNA fragments (150–250 bp) were selected for library construction and sequencing libraries were prepared using the NEXTflex™ ChIP-Seq Kit (Bioo Scientific). The libraries were sequenced using the Illumina HiSeq 2000. All the experiment has two repeats. TruSeq RNA sample prep kit protocol (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM3674392