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| Status |
Public on Aug 17, 2021 |
| Title |
TF_4_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: myc-tagging cells
chip-exo antibody: c-myc(9E10) mouse monoclonal IgG1,sc-40, lot#J2413, Santa Cruz Biotechnology
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| Growth protocol |
glycerol stocks of E. coli myc-tagged strains were inoculated into M9 minimal media with 0.2% (w/v) glucose. The culture was incubated at 37 degree 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 degree 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 TFs-DNA complexes, and then were isolated with myc antibody.
The experiments were performed following the procedures: to identify each TF binding maps in vivo, we isolated the DNA bound to each TF 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). ChIP materials (chromatin-beads) were used to perform on-bead enzymatic reactions. 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 65 degree. RNAs- and Proteins-removed DNA samples were used to perform primer extension and second adaptor ligation. The DNA samples incubated for primer extension 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. The experiments were performed in biological duplicate.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
library strategy: ChIP-exo
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 the study were mapped onto each reference genome using bowtie with default options to generate SAM output files
MACE software (https://code.google.com/p/chip-exo/) was used to detect peaks with aligned output from bowtie mapping.
Read count was calculated for each genomic position from sequence alignment, and 5% strongest intensity was used as background intensity. For each peak deteced with MACE, binding intensity was calculated by averaging read counts from two biological replicates and dividing by background intensity.
Genome_build: the reference genome ( MG1655 = NC_000913.3 )
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 |
Aug 16, 2021 |
| 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 (2) |
| GSE182078 |
A chromatin immunoprecipitation database for prokarytic organisms [E. coli K-12 MG1655 mixTF] |
| GSE182079 |
A chromatin immunoprecipitation database for prokarytic organisms |
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| Relations |
| BioSample |
SAMN20818918 |
| SRA |
SRX11788577 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5525422_TF_4_2.gff.gz |
40.4 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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