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Sample GSM3933395

Query DataSets for GSM3933395

Status

Public on Nov 25, 2019

Title

Total-Input_CtrA_C.crescentus_(AIYT-24)

Sample type

SRA

Source name

Caulobacter crescentus NA1000 culture

Organism

Caulobacter vibrioides NA1000

Characteristics

strain: NA1000
genotype: Wild type
tissue: Caulobacter crescentus NA1000 culture

Treatment protocol

C. crescentus wildtype and mutant cells were treated with formaldehyde (1% final concentration) in 1 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.

Growth protocol

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

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 rabbit polyclonal antibodies targeting CtrA (Sanselicio and Viollier,2015) (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.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Base calling: HiSeq Control Software 2.2.38, RTA 1.18.61.0, CASAVA-1.8.2
Alignment: Map with Bowtie for illumina (Galaxy Version 1.2.3), SAM-to-BAM (Galaxy Version 2.1.1)
Peak calling and refinement: MACS2 Callpeak (Galaxy Version 2.1.1.20160309.6)
Peak annotation: SeqMonk Software v1.45.1 (Braham Bionformatics Institute)
Genome_build: Caulobacter crescentus : NC_011916.1
Supplementary_files_format_and_content: The xls file reporting CtrA ChIP-Seq peaks in WT and mutants in exponential and stationary phase (as described in the paper) was generated by MACS2 software. 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).

Submission date

Jul 09, 2019

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