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| Status |
Public on Aug 12, 2020 |
| Title |
SigH (30 ℃) mock IgG rep1 |
| Sample type |
SRA |
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| Source name |
SigH (30 ℃) mock IgG rep1
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
chip antibody: Normal IgG (Millipore) and Dynabeads Pan mouse IgG (Invitrogen)
substrain: MG1655
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| Treatment protocol |
Then, cells were diluted to 1:100 into fresh same minimal medium and cultured at 30 ℃ up to mid-exponential phase (OD600 = 0.6). To treat the heat shock, cultured cells were subsequently mixed slowly with pre-warmed 55 ℃ media of equivalent volume to adjust to 42 ℃. For non-heat shocked condition, all experiments were performed in same manner except using 30 ℃ instead of 55 ℃ media. All mixture were further incubated shaking at 30 ℃ and 42 ℃ for 5 min.
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| Growth protocol |
Only in ChIP-assay for σ32, E. coli K12 MG1655 rpoH::8Myc::Kan (MG1655_rpoHmyc) isogenic strain was used, which was constructed as replacing rpoH to 8myc-rpoH encoding eight copies of the c-myc epitope tag and kan at the 3’ end. And two types of cells (50 ml) were inoculated into M9 minimal media of 5 ml supplemented 0.2% glucose, and grown at 30 ℃ with constant agitation overnight.
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
SigH30bs_exo_jMRSP_Subtr.gff
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| Data processing |
Basecalls performed using CASAVA version 1.4
All sequencing reads were mapped to E. coli MG1655 reference genome (NC_000913) using CLC Genomics Workbench5 with the length fraction of 0.9 and the similarity of 0.99.
To capture target protein binding sites corresponding genomic position of mapped reads start position (MRSP) was counted and stored for visual inspection using in-house scripts.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: gff file is generated by in-house script
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| Submission date |
Aug 22, 2014 |
| Last update date |
Aug 12, 2020 |
| Contact name |
Byung-Kwan Cho |
| Organization name |
KAIST
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| Lab |
Systems and Synthetic Biology Lab
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| Street address |
291 Daehak-ro
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| City |
Daejeon |
| ZIP/Postal code |
305-701 |
| Country |
South Korea |
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| Platform ID |
GPL17439 |
| Series (2) |
| GSE60667 |
In vivo probing of promoter overlapping between σ-factors in Escherichia coli (ChIP-exo) |
| GSE60669 |
In vivo probing of promoter overlapping between ?-factors in Escherichia coli |
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| Relations |
| BioSample |
SAMN03002756 |
| SRA |
SRX685345 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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