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| Status |
Public on Feb 19, 2015 |
| Title |
ΔbolA |
| Sample type |
SRA |
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| Source name |
bacterial cell culture
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| Organism |
Escherichia coli |
| Characteristics |
strain: no tag
genotype: ΔbolA
chip antibody: anti-Flag (Sigma)
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| Treatment protocol |
10 mM of sodium phosphate and formaldehyde to a final concentration of 1% were then added. After 10min of incubation at room temperature, the samples were incubated 30 min in ice
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| Growth protocol |
The ΔbolA and wt 3xflag strains were used to perform ChIP-seq experiments. Overnight cultures were diluted 1/100 in fresh LB medium and grown until OD600 0.6
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linked cells were harvested and washed two times with ice-cold PBS. Cells were resuspended in 450μl of TES buffer (50mM Tris-HCl pH 7.5, 150mM NaCl) and 20μl of lysis solution (13.6mg/ml lysozyme, 50% glycerol, 50mM Tris-HCl pH7.5, 100mM NaCl, 1mM DTT, 0.1% Triton X-100) followed of a 5min incubation at room temperature. 10μl of cOmplete EDTA free protease inhibitor (Roche) were added and incubated 10min at room temperature. 550μl of ChIP buffer (1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, 167mM NaCl, 20μl/ml of cOmplete EDTA free protease inhibitor (Roche) were added followed of 10min incubation at 37ºC. The lysate was then sonicated (UP200S-Hielscher) to an average size between 300bp and 700bp with 5 cycles of and amplitude of 55%, 0.45sec pulse during 10sec and 50sec in ice. Insoluble cell debris were removed by centrifugation at 20000g for 3min at 4ºC and the supernatant collected. The supernatant was added to α-Flag-agarose beads (Sigma) and incubated at 4ºC under rotation. The samples were then washed once with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl), once with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 500mM NaCl), once with LiCl wash buffer (250mM LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1), and twice with TE buffer (10mM Tris-HCl pH 8.1, 1mM EDTA). Two washes with 250μl of freshly prepared elution buffer (1% SDS, 100mM NaHCO3) were done followed by vortexing and incubation under rotation at room temperature for 15min. To reverse the cross-link 30μl of 5M NaCl were added to the elute and incubated over night at 65ºC. Finally, 4μl of 0.5M EDTA, 20μl of 1M Tris-HCl pH 6.5 and 2μl of 10mg/ml Proteinase-K (Sigma) were added and the suspension incubated at 45ºC for 2h. DNA was purified and recovered by standard phenol-chloroform extraction and ethanol precipitation with 20μg of glycogen.
Library construction was performed by processing in vitro samples to generate a library of short inserts (the DNA Colonies Template Library) by Fasteris SA, Switzerland
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Immunoprecipitated DNA
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| Data processing |
Mapping was performed using BWA tool (version 0.5.9) against the reference genome
Reference genome used: Escherichia coli K12 MG1655, version: iGenome
Peak detection and count of coverage were done using SEQMONK version 0.21.0 by Fasteris SA
Genome_build: Reference genome used: Escherichia coli K12 MG1655, version: iGenome
Supplementary_files_format_and_content: .txt file file with raw and normalized counts of coverage
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| Submission date |
Jun 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Cecilia M. Arraiano |
| E-mail(s) |
cecilia@itqb.unl.pt
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| Phone |
+351214469547
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| Organization name |
Instituto de Tecnologia Quimica e Biologica (ITQB) / Univ. Nova de Lisboa
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| Street address |
Av. Republica, Apt 127
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| City |
Oeiras |
| ZIP/Postal code |
2781-901 |
| Country |
Portugal |
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| Platform ID |
GPL14548 |
| Series (1) |
| GSE58623 |
BolA is a transcriptional switch that turns off motility and turns on biofilm development (ChIP-seq) |
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| Relations |
| BioSample |
SAMN02866192 |
| SRA |
SRX610439 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1415557_2013-06-20_HAA-2_Ecoli_K12_MG1655_WindowsCovNorm_closestGene.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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