GSM2829282: RpoB WT 2; Escherichia coli; ChIP-Seq
Crosslinked cells were then resuspended in 500 ul of lysis buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA) with 40 ul of protease inhibitor cocktail (50 mg in 0.25 ml of DMSO and 0.75 ml of TDW). Cells were lyzed with 1 ul of lysozyme for 30 min at 37oC on a rocker. 0.55 ml of 2X IP buffer (100 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2% Triton X-100 and 1 mM EDTA) were added to the sample, and then was sonicated to fragmentize genomic DNA. 0.3 ml of Wash buffer I (50 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1% Triton X-100 and 1mM EDTA) was added to make the volume up to 1.4 ml. Only 0.7 ml was taken and transfered to a new tube, and 10 ul of Anti-rpoB mouse antibody was added, and the sample was incubated overnight at 4 oC to make Antibody-TF complex. 50 ul of Dynabeads Pan mouse IgG were washed 3 times with bead washing solution (250 mg BSA in 50 ml of PBS), and were added to the sample. Cell lysate with beads were incubated for 6 hours or overnight at 4oC to make Dynabead-antibody-TF complex. The beads were pulled down on a magnet stand, and washed 2 times with wash buffer I and with wash buffer II (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1% Triton X-100 and 1mM EDTA), wash buffer III (10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1% Triton X-100 and 1mM EDTA), and wash buffer IV (10 mM Tris-HCl (pH 8.0), 1mM EDTA). The bead-bound TF-DNA complex was then end-repaired, dA-tailed, and ligated to the first adapter. Adapter-ligated sample was then treated with nick-repair reagent, and was treated with lambda exonuclease and RecJ exonuclease. Then DNA was eluted away from Dynabeads by incubating in 200 ul of elution buffer (50 mM Tris-HCl (pH 8.0), 1% SDS and 1 mM EDTA) at 65oC overnight. Protein was removed by treating 4 ul of protease K and being incubated at 55 oC for 2 hours, and by Phenol-Chloroform-IAA extraction. Purified DNA was used to bulid the second strand synthesis, followed by another dA-tailing, second strand ligation, and 3' overhang removal stpes. Then the sequencing library was amplified with PCR enrichment. ChIP-seq libraries were prepared for sequencing using standard Illumina protocols
GEO
GSM2829282
GEO Accession
GSM2829282