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Sample GSM2516006

Query DataSets for GSM2516006

Status

Public on Nov 01, 2017

Title

ChIP-Seq_OpaA_NA1000+Rif

Sample type

SRA

Source name

cultures

Organism

Caulobacter vibrioides

Characteristics

strain: NA1000
genotype: Wild type
antibody: antibody was raised in rabbits against purified OpaA protein

Growth protocol

Overnight saturated cultures of each Caulobacter crescentus strains were freshly diluted in PYE rich media (O.D.660nm~0.025) and cultures were grown at 30°C with aeration to mid-log phase (O.D.660nm~0.5).

Extracted molecule

genomic DNA

Extraction protocol

Overnight saturated cultures of each C. crescentus strains were freshly diluted in PYE rich media (O.D.660nm~0.025) and cultures were grown at 30°C with aeration to mid-log phase (O.D.660nm~0.5). Then, after 10 min of antibiotic treatments (Rifampicin 30µg/ml; Novobiocin 100µg/ml; Chloramphenicol 1µg/ml) or without, cells were cross-linked in presence of 1% of formaldehyde and 10 μM of sodium phosphate (pH 7.6) during 10 minutes at room temperature and thereafter on ice for 30 min, then washed three times in phosphate buffered saline (PBS), re-suspended in TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl) containing 10 mM of DTT and incubated during 10 minutes at 37°C after the addition of Ready-Lyse lysozyme solution (Epicentre, Madison, WI), according to the manufacturer's instructions. Lysates were then sonicated (Bioruptor® Pico, www.diagenode.com) 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. Lysates were then 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, www.roche.com) 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 reserved as input sample. Then, the pre-cleared supernatants were incubated overnight at 4°C with OpaA Polyclonal antibodies (1:1,000 dilution). The immuno-complexes were captured after incubation with Protein-A agarose pre-saturated with BSA during 2 hours at 4°C and washed once 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), then once 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), once with LiCl washing buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and twice with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA). The complexes were eluted twice with 250 μL of elution buffer (SDS 1%, 0.1 M NaHCO3, freshly prepared) and incubated overnight at 65°C with 300 mM NaCl to reverse the crosslinks. The samples were then treated with 2 µg of Proteinase K for 2 hours 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 100 μl of 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.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Data processing

Base calling: HiSeq Control Software 1.5.15.1, RTA 1.13.48.0, CASAVA-1.8.2
Alignment: Bowtie-0.12.9, samtools-0.1.18
Peak calling and refinement : MACS2 Software (Galaxy Version 2.1.0.20151222.0)
Peak annotation: SeqMonk Software v0.34.1 (Braham Bionformatics Institute)
Genome_build: Caulobacter crescentus : NC_011916.1
Supplementary_files_format_and_content: The xls file reporting OpaA 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: Closest CDS; column 11: GC% (+/-50pb window surrounding the peak summit) and column 12: DNA sequences (+/-75pb window surrounding the peak summit).

Submission date

Feb 28, 2017

Last update date

May 01, 2023

Contact name

Patrick H. Viollier

E-mail(s)

[email protected]

Organization name

University of Geneva, Faculty of Medicine / CMU

Department

Dept. Microbiology and Molecular Medicine