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| Status |
Public on Dec 23, 2016 |
| Title |
popZ-KE_ZitP-N |
| Sample type |
SRA |
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| Source name |
Caulobacter crescentus
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| Organism |
Caulobacter vibrioides |
| Characteristics |
strain: NA1000
genotype: popZ-mCherry(KE)
antibody: α-ZitP(N-TER) antibody
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| Treatment protocol |
none
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| Growth protocol |
Caulobacter crescentus and derivatives were grown at 30°C in PYE (Peptone yeast extract).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mid-log phase cells were cross-linked in 10 mM sodium phosphate (pH 7.6) and 1% formaldehyde at room temperature for 10 min and on ice for 30 min thereafter, washed thrice in phosphate buffered saline (PBS) and lysed in a Ready-Lyse lysozyme solution (Epicentre, Madison, WI) according to the manufacturer`s instructions. Lysates were sonicated (Sonifier Cell Disruptor B-30; Branson Sonic Power. Co., Danbury, CT) on ice using 10 bursts of 20 sec at output level 4.5 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. Lysates were normalized by protein content, diluted to 1 mL using ChIP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl plus protease inhibitors (Roche) and pre-cleared with 80 μL of protein-A agarose (Roche, Switzerland) and 100 uμg BSA. Ten % of the supernatant was removed and used as total chromatin input DNA as described before (Radhakrishnan et al., 2008). Two microliters of polyclonal antibodies were added to the remains of the supernatant, incubated overnight at 4°C with 80 μL of protein-A agarose beads pre saturated with BSA, washed once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl) and LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1) and twice with TE buffer (10 mM Tris-HCl (pH 8.1) and 1 mM EDTA). The protein•DNA complexes were eluted in 500 μL freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3), supplemented with NaCl to a final concentration of 300 mM and incubated overnight at 65°C to reverse the crosslinks. The samples were 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 extracted using phenol:chloroform:453 isoamyl alcohol (25:24:1), ethanol-precipitated using 20 ug glycogen as carrier and re-suspended in 100uL of water. For deep sequencing (ChIP-seq) total chromatin input DNA was not saved.
DNA libraries were prepared for sequencing using standard Illumina protocols by FASTERIS SA, Switzerland
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Base calling: HiSeq Control Software 2.0.12.0, RTA 1.17.21.3, CASAVA-1.8.2
Alignment: BWA, samtools (via Galaxy plattform)
Peak calling: SeqMonk software (Braham Bionformatics Institute)
Genome_build: Caulobacter crescentus : NC_011916.1;
Supplementary_files_format_and_content: The processed data files report the coverage, expressed as a percentage of reads per probe, for the peak region covering the origin of replication of Caulobacter crescentus.Column1: location of the peak; column 2 percentage of reads per probe
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| Submission date |
Dec 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Antonio Frandi |
| E-mail(s) |
[email protected]
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| Organization name |
University of Geneva
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| Department |
Microbiology and Molecular Medicine
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| Lab |
Patrick Viollier Lab
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| Street address |
rue michel servet 1
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| City |
Geneva |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
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| Platform ID |
GPL21693 |
| Series (1) |
| GSE79918 |
Ancestral (bi-)polarization control module in free-living and obligate intracellular bacteria |
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| Relations |
| BioSample |
SAMN06101800 |
| SRA |
SRX2391054 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2413408_GHA618.txt.gz |
847 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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