GSM1314507: RNAP (+Rif) ChIP-seq; Escherichia coli str. K-12 substr. MG1655; ChIP-Seq
Cultured cells (50 mL) were cross-linked by 1% formaldehyde at room temperature for 30 min, followed by adding 2 mL of 2.5 M glycine for the quenching of the unused formaldehyde. After washing three times with 50 mL of ice-cold Tris-buffered saline (TBS), the washed cells were resuspended in 0.5 mL of lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 μg/mL RNaseA, protease inhibitor cocktail and 1 kU Ready-Lyse lysozyme (Epicentre, Madison, WI) and incubated at 37 oC for 30 min. The cells were then treated with 0.5 mL of 2×IP buffer (100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 2%(v/v) Triton X-100, and protease inhibitor cocktail), followed by incubation on ice for 30 min. The lysate was then sonicated in an ice bath to fragment the chromatin complexes using Sonic Dismembrator Model 500 (four times for 20 s each, output level, 2.5). After removing cell debris by centrifugation, the size distribution of fragmented DNA in the resulting supernatant was confirmed using agarose gel electrophoresis (200-400 bp). After stringent sequential washings, the IP-DNAs were reverse-crosslinked from DNA-protein complexes at 65 oC overnight. The RNAs and proteins in the supernatant were degraded with 1 µg RNase A and 8 µg protease K, respectively. The IP-DNAs were further purified using Qiagen PCR purification kit. The purified IP-DNAs were then modified and amplified by TruSeq DNA Sample Prep Kit v2 for the Illumina next-generation sequencing according to the manufacturer’s instruction (Illumina). The PCR-amplified DNA products were then loaded onto 2% agarose gel and extracted using QIAquick gel purification columns.
GEO Accession
GSM1314507