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| Status |
Public on Mar 29, 2020 |
| Title |
ChIP-Seq_GcrA(Totalinput)_H.neptuniumLE670_(GHA-466) |
| Sample type |
SRA |
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| Source name |
ChIP-Seq_GcrA(Totalinput)_H.neptuniumLE670_(GHA-466)
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| Organism |
Hyphomonas neptunium |
| Characteristics |
strain: LE670 (ATCC15444)
genotype: Wild type
antibody: input
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| Treatment protocol |
H. neptunium wildtype cells were treated with formaldehyde (1% final concentration) in 10 uM sodium phosphate buffer (pH 7.6) at RT for 10 min to achieve crosslinking. Subsequently, the cultures were incubated for an additional 30 min on ice and washed three times in phosphate buffered saline (PBS, pH 7.4). The resulting cell pellets were frozen in liquid nitrogen and stored at -80°C.
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| Growth protocol |
Overnight saturated cultures of Hyphomonas neptunium LE670 strains were freshly diluted in Difco Marine Broth 2216 media and cultures were grown at 28°C with aeration to mid-log phase (O.D.580nm~0.5).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After resuspension of the cells in TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl) containing 10 mM of DTT, they were incubated in the presence of Ready-Lyse lysozyme solution (Epicentre, Madison, WI) for 10 minutes at 37°C, according to the manufacturer's instructions. Lysates were sonicated (Bioruptor® Pico) at 4°C using 15 bursts of 30 sec to shear DNA fragments to an average length of 0.3-0.5 kbp and cleared by centrifugation at 14,000 rpm for 2 min at 4°C. The volume of the lysates was then adjusted (relative to the protein concentration) to 1 ml using ChIP buffer (0.01% SDS, 1.1% Triton X-84 100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl) containing protease inhibitors (Roche) and pre-cleared with 80 μl of protein-A agarose (Roche, www.roche.com) and 100 μg BSA. 2% of each pre-cleared lysate was kept as total input samples (negative control samples). The rest of the pre-cleared lysates was incubated overnight at 4°C with polyclonal antibodies targeting CtrAHN (this work) or GcrACC (Holtzendorff et al., 2004) (1:1,000 dilution). The immuno-complexes were captured after incubation with Protein-A agarose (pre-saturated with BSA) during a 2 h incubation at 4°C and they were washed subsequently with low salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), with high salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), with LiCl washing buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and finally twice with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA). The complexes were eluted from the Protein-A agarose beads with two times 250 μl elution buffer (SDS 1%, 0.1 M NaHCO3, freshly prepared) and then, just like total input samples, incubated overnight with 300 mM NaCl at 65°C to reverse the crosslinks. The samples were then treated with 2 μg of Proteinase K for 2 h at 45°C in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5). DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1), ethanol-precipitated using 20 μg of glycogen as a carrier and resuspended in 50 μl of DNAse/RNAse free water.
Immunoprecipitated chromatin was used to prepare sample libraries used for deep-sequencing at Fasteris SA (Geneva, Switzerland). ChIP-Seq libraries were prepared using the DNA Sample Prep Kit (Illumina) following manufacturer instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Base calling: HiSeq Control Software 2.2.38, RTA 1.18.61.0, CASAVA-1.8.2
Alignment: Bowtie-0.12.9, samtools-0.1.18
Peak calling and refinement: MACS2 Software (Galaxy Version 2.1.1.20160309.0)
Peak annotation: SeqMonk Software v1.40.0 (Braham Bionformatics Institute)
Genome_build: Hyphomonas neptunium LE670 (ATCC15444) : NC_008358
Supplementary_files_format_and_content: The xls file reporting CtrA and GcrA ChIP-Seq peaks (described in the paper) was generated by MACS2 and SeqMonk softwares. The sheet is organized in columns as follows: column 1: Peak # (order on chromosome); column 2: Peak start coordinates (bp); column 3: Peak end coordinates (bp); column 4: Peak length coordinates (bp); column 5: Peak summit coordinates (bp); column 6: Peak pileup; column 7: -LOG10(pvalue); column 8: Fold enrichment; column 9: -LOG10(qvalue); column 10: DNA sequences (+/-75pb window surrounding the peak summit).
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| Submission date |
Jan 11, 2019 |
| Last update date |
May 01, 2023 |
| Contact name |
Patrick H. Viollier |
| E-mail(s) |
[email protected]
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| Organization name |
University of Geneva, Faculty of Medicine / CMU
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| Department |
Dept. Microbiology and Molecular Medicine
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| Street address |
Rue Michel Servet 1
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| City |
Geneva 4 |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
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| Platform ID |
GPL26030 |
| Series (2) |
| GSE124953 |
Integrative and quantitative analysis reveals proper CtrA-dependent cell cycle regulation in the absence of the DivJ/PleC asymmetry module in the stalked budding bacterium Hyphomonas neptunium |
| GSE134367 |
Integrative and quantitative view of the CtrA regulatory network in a stalked budding bacterium |
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| Relations |
| BioSample |
SAMN10724769 |
| SRA |
SRX5242118 |
| 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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