title: GSM1482120: ArgR (+arg) rep1 and rep2; Escherichia coli str. K-12 substr. MG1655; ChIP-Seq
library_construction_protocol: Cultured cells (50 mL) were cross-linked with 1% formaldehyde at room temperature for 30 min and added 2 mL of 2.5M glycine to quench 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 37oC 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 using Sonic Dismembrator Model 500 (four times for 20 s each, output level, 2.5). Size distribution of the fragmented DNAs was confirmed using agarose gel electrophoresis (200-400 bp) after removing cell debris by centrifugation. The cross-linked DNA-ArgR complexes in the supernatant were then immunoprecipitated by adding 10 µL of Anti-myc (9E10) (Santa Cruz, Dallas, TX). For mock-IP control, 2 µg of normal mouse IgG (Santa Cruz) was added into the supernatant in parallel. They were then incubated overnight at 4oC with constant rotation. The cross-linked DNA-protein and antibody complexes were selectively captured by adding 50 µL of Dynabeads Pan Mouse IgG magnetic beads (Invitrogen, Grand Island, NY). Then, DNAs 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 previously. Then, protein-DNA complexes were reverse-cross-linked by heating at 65°C overnight and proteins were degraded by 8 µg of protease K (Invitrogen). The purified DNAs were denatured at 95°C and extended by P1 primer (5’-OH-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT), further 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.
attributes.tag: GEO Accession
attributes.value: GSM1482120