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| Status |
Public on Aug 17, 2021 |
| Title |
rpoD myc-tag 2 |
| Sample type |
SRA |
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| Source name |
bacteria cells
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| Organism |
Escherichia coli K-12 |
| Characteristics |
strain: MG1655
myc-tagging: wild type cells
chip-exo antibody: purified anti E.coli sigma factor 70, clone 2G10, BioLegend
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| Treatment protocol |
For regulatory condition, M9 minimal media was supplemented with 1 mL trace element solution (100X) containing 1 g EDTA, 29 mg ZnSO4.7H2O, 198 mg MnCl2.4H2O, 254 mg CoCl2.6H2O, 13.4 mg CuCl2, and 147 mg CaCl2.
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| Growth protocol |
Glycerol stocks of E. coli strains were inoculated into M9 minimal media with 0.2% (w/v) glucose. The culture was incubated at 37 oC overnight with agitation, and then was used to inoculate the fresh media (1/200 dilution). The volume of the fresh media was 150 mL for each biological replicate. The fresh culture was incubated at 37 oC with agitation to the mid-log phase (OD600 ≈ 0.5).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated uncharacterized transcripton factors-DNA complexes were isolated with myc antibody.
ChIP-exo experiment was performed following the procedures: to identify each TF candidate binding maps in vivo, we isolated the DNA bound to each TF candidate from formaldehyde cross-linked E. coli cells by chromatin immunoprecipitation (ChIP) with the specific antibodies that specifically recognizes myc tag (9E10, Santa Cruz Biotechnology), and Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) followed by stringent washings as described previously [42]. ChIP materials (chromatin-beads) were used to perform on-bead enzymatic reactions of the ChIP-exo method [11]. Briefly, the sheared DNA of chromatin-beads was repaired by the NEBNext End Repair Module (New England Biolabs) followed by the addition of a single dA overhang and ligation of the first adaptor (5’-phosphorylated) using dA-Tailing Module (New England Biolabs) and NEBNext Quick Ligation Module (New England Biolabs), respectively. Nick repair was performed by using PreCR Repair Mix (New England Biolabs). Lambda exonuclease- and RecJf exonuclease-treated chromatin was eluted from the beads and the protein-DNA cross-link was reversed by overnight incubation at 65oC. RNAs- and Proteins-removed DNA samples were used to perform primer extension and second adaptor ligation with following modifications. The DNA samples incubated for primer extension as described previously [12] were treated with dA-Tailing Module (New England Biolabs) and NEBNext Quick Ligation Module (New England Biolabs) for second adaptor ligation. The DNA sample purified by GeneRead Size Selection Kit (Qiagen) was enriched by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The amplified DNA samples were purified again by GeneRead Size Selection Kit (Qiagen) and quantified using Qubit dsDNA HS Assay Kit (Life Technologies). Quality of the DNA sample was checked by running Agilent High Sensitivity DNA Kit using Agilent 2100 Bioanalyzer (Agilent) before sequenced using HiSeq (Illumina) in accordance with the manufacturer’s instructions. Each modified step was also performed in accordance with the manufacturer’s instructions. ChIP-exo experiments were performed in biological duplicate.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
The base calling was done using Illumina’s Real-Time Analysis (RTA) v2.7.7; data was converted to fastq and demultiplexed using Illumina’s bcl2fastq2 Conversion Software v2.20;
Sequence reads generated from ChIP-exo were mapped onto the each reference genome using bowtie with default options to generate SAM output files
MACE program was used to define peak candidates from biological duplicates for each experimental condition with sequence depth normalization.
To reduce false-positive peaks, manual curation was performed based on the signal-to-noise(S/N) ratio value.
The calculation of S/N ratio resembles the way to calculate ChIP-chip peak intensity where IP signal was divided by Mock signal. Then, each peak was assigned to the nearest gene.
Genome_build: The reference genome MG1655 = NC_000913.2
Supplementary_files_format_and_content: Tab-delimited text files in gff format which has 8 columns: sequence id, source(empty), feature (+/- strand), start position, end position, intensity score, strand(+/-), frame(.), attribute(.).
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| Submission date |
Oct 21, 2020 |
| Last update date |
Aug 17, 2021 |
| Contact name |
Ye Gao |
| E-mail(s) |
[email protected]
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| Organization name |
UCSD
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| Street address |
9500 Gilman Dr.
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| City |
La Jolla |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL24570 |
| Series (1) |
| GSE159777 |
ChIP-exo analysis reveals novel uncharacterized transcription factors involved in the diverse regulatory functions of Escherichia coli |
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| Relations |
| BioSample |
SAMN16509803 |
| SRA |
SRX9333773 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4844462_mg1655_rpoD2.gff.gz |
33.7 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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