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| Status |
Public on May 25, 2014 |
| Title |
CtrA_∆pleC_ChIPseq |
| Sample type |
SRA |
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| Source name |
Mid-log phase cells
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| Organism |
Caulobacter vibrioides |
| Characteristics |
strain: NA1000
genotype: ∆pleC
antibody: anti-Cc_CtrA
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| Growth protocol |
Caulobacter crescentus and derivatives were grown at 30°C in PYE (Peptone yeast extract) or M2G, while Sinorhizobium fredii NGR234 was grown at 30°C in TY (Triptone 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 to CtrA (Domian et al., 1997), SciP (Gora et al., 2010; Tan et al., 2010) (note that for ChIP experiments reported in Table S6 we used antibodies that we raised against His6-SUMO-SciP), FlbD (Davis and Viollier, 2011), RpoC (Neoclone, Madison, WI), MucR1 or MucR2 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 HiSeq 2500 |
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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: Bowtie-0.12.9, samtools-0.1.18
Peak calling: SeqMonk software (Braham Bionformatics Institute)
Peak refinement and annotation: SeqMonK output were processed by Java and Perl programs (in house written, decribed in the paper) to refine the peaks annotation and eliminate background
ChIPseq comparisons: Peaks list after being proccessed were scanned for common target, then ratio of each peak abundance were computed and reported as the log2 fold variation
Genome_build: Caulobacter crescentus : NC_011916.1; Sinorhizobium fredii : NC_012587.1, NC_000914.2, NC_012586.1
Supplementary_files_format_and_content: the xls files reporting peaks annotations and abundance were generated by Java and Perl programs (described in the paper) and consist of two sheet the ANNO and the NO-ANNO sheet. The ANNO probes sheets are organized in columns as follows: column 1: gene name to which the peak is associated is reported in the reference genome used for the reads alignment column 2 : probe start column 3 : probe end column 4 : COG categories to which the gene associated to the corresponding peak belongs to column 5 : PubMed Identification number of the protein encoded by the gene associated to the peak column 6 : Length of the protein column 7 : The gene strand of the gene associated to the peak (0 plus strand, 1 reverse strand) column 8 : Gene start column 9 : Gene end column10: Probe distance, the distance of the probe from ORF start column 11 : It is reported here the abundance of reads per probe expressed as the percentage of total reads number column 12: N possible association, it is reported here the number of possible association that the probe associated to the gene reported in the first column can have (i.e.: two genes that are divergent but shares the same peak) column 13 : Name of the gene that can be associated to this probe and the distance of the probe from the gene start) The NO-ANNO probes files contain all those probes that made the cut off, (are recognized as peaks) but they cannot be associated to any genes. File organization is the same as before except that only information about the probe location and reads abundance are reported.
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| Submission date |
Dec 01, 2013 |
| 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 |
GPL18006 |
| Series (1) |
| GSE52849 |
Cell cycle transition from S-phase to G1 in Caulobacter is mediated by ancestral virulence regulators |
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| Relations |
| BioSample |
SAMN02435751 |
| SRA |
SRX387070 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1277026_CtrA_delta_pleC_peaks.xls.gz |
36.3 Kb |
(ftp)(http) |
XLS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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