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Sample GSM7927866

Query DataSets for GSM7927866

Status

Public on May 10, 2024

Title

Total-Input_CdnL_Caulobacter_crescentus_NA1000_ΔcdnL

Sample type

SRA

Source name

NA1000

Organism

Caulobacter vibrioides

Characteristics

strain: NA1000
genotype: delta-cdnL

Treatment protocol

Cells were harvested (centrifugation at 8000 rpm during 10 min at 25°C) and, cell pellets were washed 3 times with 50 ml of M2 media (without glucose) at 25°C. Each of the washed cell pellets was split into two and used to inoculate 50 ml of M2 (carbon starvation) and 50 ml of M2G (preheated culture medium at 30°C), respectively. Cultures were incubated at 30⁰C (shaking at 200 rpm) for additional 60 minutes and then, supplemented with 10 μM sodium phosphate buffer (pH 7.6) and treated with formaldehyde (1% final concentration) 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 stored at -80°C.

Growth protocol

C. crescentus strains WT (EG865), ∆cdnL (EG1898) and CdnLDD (EG2530) were grown overnight in 6 ml M2G (M2 with 0.2% glucose w/v) at 30°C (shaking at 200 rpm), respectively. The next day, cultures were diluted into 100 mL M2G and grown to an OD660nm of 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, the cell resuspensions 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. Five percent of the pre-cleared lysates were kept as total input samples. The rest of the pre-cleared lysates were then incubated overnight at 4°C with polyclonal rabbit antibodies targeting CdnL (1:1000 dilution). The Immuno-complexes were captured by incubation with Protein-A agarose beads (pre-saturated with BSA) during a 2 h incubation at 4°C and then, 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 immuno-complexes were eluted from the Protein-A beads with two times 250 μL elution buffer (SDS 1%, 0.1 M NaHCO3, freshly prepared) and then, just like the total input sample, 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 30 μl of DNAse/RNAse free water.
Immunoprecipitated chromatins were 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. Single-end run was performed on an Illumina Next-Generation DNA sequencing instruments (NextSeq High), 50 cycles were performed and yielded several million reads per sequenced samples.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

Base calling: NextSeq Control Software 4.0.2.7, RTA 2.11.3, bcl2fastq2.17 v2.17.1.14
Read Quality: FastQC (Galaxy Version 0.74+galaxy0), FASTQ Trimmer (Galaxy Version 1.1.5)
Alignment: Bowtie2 (Galaxy Version 2.4.5+galaxy1)
Peak calling: MACS2 (Galaxy Version 2.2.7.1+galaxy0 , No broad regions option) relative to the ΔcdnL negative ChIP-seq control. The q-value (false discovery rate, FDR) cut-off for called peaks was 0.05.
ChIP-Seq profiles RPM normalized and annotation: SeqMonk Software v1.47.2 (Braham Bionformatics Institute)
Assembly: Caulobacter crescentus NA1000 (NC_011916.1)
Supplementary files format and content: The xlsx file reporting CdnL (WT and *DD mutant) ChIP-Seq RPM normalized profiles was generated by SeqMonk software. The sheet is organized in columns as follows: column 1: Start (bp); column 2: End (bp); column 3: Feature; column 4: Description; column 5: Feature Strand; column 6: Feature Type; columns 7 to 15: CdnL (WT and *DD mutant) plus controls ChIP-Seq profiles (normalized Read Per Million unit, RPM)

Submission date

Dec 01, 2023

Last update date

May 10, 2024

Contact name

Patrick H. Viollier

E-mail(s)

Patrick.Viollier@unige.ch

Organization name

University of Geneva, Faculty of Medicine / CMU

Department

Dept. Microbiology and Molecular Medicine