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| Status |
Public on Jan 13, 2021 |
| Title |
mock by IgG for RNAP-Rif rep1 |
| Sample type |
SRA |
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| Source name |
mock by IgG for RNAP-Rif
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
chip antibody: Normal IgG [Millipore, catalog# 12-371, lot# DAM1774722] and Dynabeads Pan mouse IgG [Invitrogen, catalog# 11041, lot# 131306200]
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| Treatment protocol |
For rifampicin-treated cells, rifampicin dissolved in methanol was added to a final concentration of 150 μg/mL with subsequent stirring for 30 min. Cell growth was monitored at OD600 nm to verify the inhibitory effects of rifampicin.
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| Growth protocol |
Glycerol stocks of E. coli K12 strain MG1655 were inoculated into M9 minimal media supplemented 0.2% glucose and grown at 30 oC with constant agitation overnight. Cultures were diluted 1:100 into fresh minimal medium and then cultured at 30 ℃ to mid-exponential phase (OD600 nm ~ 0.6).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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).
Prior to the reverse-crosslinking in the ChIP precedure, 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 disgested with λ exonuclease (NEB) as reported previously. 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 ℃ 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 oC 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 |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Description |
Sample 11
Processed data file(s):
RNAP_NoRif_ChIPexo.gff.gz
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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.
gff file is generated by in-house script
Genome_build: ASM584v1
Supplementary_files_format_and_content: .gff files. We followed that gff format definition of "http://www.sanger.ac.uk/resources/software/gff/spec.html" but some columns are ignored because they do not affect the visualization. In detail, fields are: <contig id> <source but ignored> <feature> <start> <end> <MRSP height> <strand> <frame ignored> [attributes] [comments]. In this, we defined MRSP (Mapped Reads Start Position) as a number of reads that are started at the genomic position. For the reads that are mapped to reverse strand, we assigned minus value for the visualization convenience. To reduces inter-library variance, we converted the value as of 1 million reads similar to RPKM. In biological meaning, MRSP indicates cross-linked sites for the ChIP-exo and putative TSS site TSS-seq and so on.
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| Submission date |
Jan 26, 2014 |
| Last update date |
Jan 13, 2021 |
| 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) |
| GSE54403 |
In vivo organization of bacterial transcription initiation complexes at a genome-scale [ChIP-exo] |
| GSE54407 |
In vivo organization of bacterial transcription initiation complexes at a genome-scale |
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| Relations |
| SRA |
SRX450241 |
| BioSample |
SAMN02596576 |
| 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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