title: GSM1484357: SigH (30 ℃); Escherichia coli str. K-12 substr. MG1655; ChIP-Seq
library_construction_protocol: Cultured cells of 50 ml were cross-linked by 1% formaldehyde at room temperature for 30 min. To quench the unused formaldehyde and to stop the reaction, 2 mL of 2.5 M glycine was added to the cell. The cells were washed three times with 50 mL of ice-cold Tris-buffered saline (TBS) and then were incubated with 0.5 mL of lysis buffer (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)) at 37 ℃ for 30 min1. 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. For the lysis of cell and fragmentation of DNA, the lysate was then sonicated in an ice bath using Sonic Dismembrator Model 500 (four times for 20 s each, output level, 2.5). The size distribution of fragmented DNAs was confirmed using agarose gel electrophoresis (200-400 bp) after removing cell debris by centrifugation. After stringent sequential washings, the IP-DNAs were reverse-crosslinked from DNA-protein complexes at 65 ℃ 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.
attributes.tag: GEO Accession
attributes.value: GSM1484357