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| Status |
Public on Apr 18, 2018 |
| Title |
Cra glucose 1 |
| Sample type |
SRA |
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| Source name |
cra-8myc tagged strain_glucose
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| Organism |
Escherichia coli |
| Characteristics |
strain: K-12 MG1655
genotype/variation: cra-8myc-tagged
cultured in: M9 minimal media with 0.2% glucose
growth phase: mid-log
chip antibody: anti-myc
chip antibody vendor: Santa Cruz Biotech
chip antibody cat. #: sc-28207
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| Growth protocol |
E. coli K-12 MG1655 cra-8myc tagged strains was grown to mid-log phase aerobically at 37°C in M9 minimal media supplemented with 0.2% glucose, fructose and acetate.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
E. coli cells were crosslinked in 1% formaldehyde for 25 minutes at RT on a rocker, then washed 3 times with 50 ml of ice-cold TBS (Tris buffered saline) each time.
Crosslinked cells were then resuspended in 500 ul of lysis buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA) with 40 ul of protease inhibitor cocktail (50 mg in 0.25 ml of DMSO and 0.75 ml of TDW). Cells were lyzed with 1 ul of lysozyme for 30 min at 37C on a rocker. 0.55 ml of 2X IP buffer (100 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2% Triton X-100 and 1 mM EDTA) were added to the sample, and then was sonicated to fragmentize genomic DNA. 0.3 ml of Wash buffer I (50 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1% Triton X-100 and 1mM EDTA) was added to make the volume up to 1.4 ml. Only 0.7 ml was taken and transfered to a new tube, and 15 ul of Anti-c-myc mouse antibody was added, and the sample was incubated overnight at 4C to make Antibody-TF complex. 50 ul of Dynabeads Pan mouse IgG were washed 3 times with bead washing solution (250 mg BSA in 50 ml of PBS), and were added to the sample. Cell lysate with beads were incubated for 6 hours or overnight at 4C to make Dynabead-antibody-TF complex. The beads were pulled down on a magnet stand, and washed 2 times with wash buffer I and with wash buffer II (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1% Triton X-100 and 1mM EDTA), wash buffer III (10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1% Triton X-100 and 1mM EDTA), and wash buffer IV (10 mM Tris-HCl (pH 8.0), 1mM EDTA). The bead-bound TF-DNA complex was then end-repaired, dA-tailed, and ligated to the first adapter. Adapter-ligated sample was then treated with nick-repair reagent, and was treated with lambda exonuclease and RecJ exonuclease. Then DNA was eluted away from Dynabeads by incubating in 200 ul of elution buffer (50 mM Tris-HCl (pH 8.0), 1% SDS and 1 mM EDTA) at 65C overnight. Protein was removed by treating 4 ul of protease K and being incubated at 55C for 2 hours, and by Phenol-Chloroform-IAA extraction. Purified DNA was used to bulid the second strand synthesis, followed by another dA-tailing, second strand ligation, and 3' overhang removal stpes. Then the sequencing library was amplified with PCR enrichment.
ChIP-seq libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Description |
Immunoprecipitated DNA
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| Data processing |
ChIP-exo reads were aligned to the ASM584v2 genome reference sequence using using bowtie v1.0.0 with parameters -S
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 95% strongest intensity was used as background intensity.
For each peak detected with MACE, binding intensity was calculated by averaging read counts from two biological replicates and dividing by background intensity.
Genome_build: ASM584v2
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 |
Feb 04, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Donghyuk Kim |
| E-mail(s) |
dkim@unist.ac.kr
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| Organization name |
UNIST
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| Department |
Department of Chemical Engineering
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| Lab |
Systems Biology Lab
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| Street address |
50 UNIST-gil, Eonyang-eup, Ulju-gun
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| City |
Ulsan |
| ZIP/Postal code |
44919 |
| Country |
South Korea |
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| Platform ID |
GPL16085 |
| Series (2) |
| GSE65641 |
Systems approach elucidates convoluted transcriptional regulation on carbon metabolism [ChIP-seq] |
| GSE65643 |
Systems approach elucidates convoluted transcriptional regulation on carbon metabolism |
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| Relations |
| BioSample |
SAMN03329440 |
| SRA |
SRX865351 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1602341_cra_glucose_1.gff.gz |
25.8 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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