GSM1484355: RNAP beta (42 ℃) rep2; Escherichia coli str. K-12 substr. MG1655; OTHER
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. Prior to the reverse-crosslinking in the ChIP procedure, the IP-DNA in the crosslinked DNA-protein complexes attached on the magnetic beads were end-polished using T4 DNA polymerase (NEB, Ipswich, MA), ligated with the annealed adaptor 1 (5’- Phospho-AACTGCCCCGGGTTGCTCTTCCGATCT and 5’- OH-AGATCGGAAGAGC-OH), nick-repaired using phi29 polymerase (NEB), and digested with λ exonuclease (NEB) as reported previously3. Between the enzymatic treatments, the crosslinked DNA-protein complexes were sequentially washed with 10 mM TE (pH 8.0) and 10 mM Tris-HCl (pH 7.5-8.0). The magnetic beads were then resuspended in 50 µL of elution buffer (50 mM Tris-HCl (pH 8.0), 1% SDS, and 1 mM EDTA). The exonuclease-treated IP-DNA (IP-exo-DNA) was then reverse-crosslinked from DNA-protein complexes at 65 oC overnight. The RNAs and proteins in the supernatant were degraded with 1 µg of RNase A (Invitrogen) and 8 µg of protease K (Invitrogen), respectively. The IP-exo-DNAs were further purified using Qiagen PCR purification kit according to the manufacturer’s instruction (QIAGEN, Hilden, Germany). The purified IP-exo-DNAs were denatured at 95 ℃ and extended by P1 primer (5’-OH-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT). Subsequently, the extended ends of the IP-exo-DNAs were ligated with the annealed adaptor 2 (5’-OH-ACACTCTTTCCCTACACGACGCTCTTCCGATCT and 5’-OH-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAG). The ligated DNA products were purified using Qiagen PCR purification kit and were PCR-amplified by P2 primer (5’-OH-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and P3 primer (5’-OH-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT). The degenerative sequence (the underlined 6Ns) in the P3 primer indicates the index sequence for the Illumina next-generation sequencing (Illumina, San Diego, CA). The PCR-amplified DNA products were then loaded onto 2% agarose gel and extracted using QIAquick gel purification columns.
GEO Accession
GSM1484355