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| Status |
Public on Nov 09, 2023 |
| Title |
ChIP-Seq_DjlA-HA_Caulobacter_crescentus_UG14074_PYE+Nal20. |
| Sample type |
SRA |
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| Source name |
UG14074
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| Organism |
Caulobacter vibrioides |
| Characteristics |
strain: UG14074
genotype: {delta}bla (CCNA_02223) xyl::Pxyl-djlA-HA
treatment: Medium supplemented with 0,3% Xylose and treated with Nalidixic acid (20µg/ml) 30 min prior fixation
chip antibody: Anti-HA (Merck Millipore, Clone114-2C-7)
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| Treatment protocol |
Cultures of exponentially growing cells (O.D.660nm of 0.5) were supplemented with 10 μM sodium phosphate buffer (pH 7.6) and then treated with formaldehyde (1% final concentration) at RT for 10 minutes to achieve crosslinking. Subsequently, the cultures were incubated for an additional 30 minutes on ice and washed three times in phosphate buffered saline (PBS, pH 7.4). The resulting cell pellets were stored at -80°C.
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| Growth protocol |
Overnight cultures in PYE of C. crescentus were freshly restarted (starting O.D.660nm~0.05) in 80ml of PYE supplemented with Xylose 0,3% and incubated at 30°C under agitation. Nalidixic acid treated culture was supplemented with 20 μg/mL of antibiotic 30 minutes before being fixed.
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| 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 seconds to shear DNA fragments to an average length of 0.2-0.5 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. 5% of each pre-cleared lysate were reserved as total input samples (negative control samples). The pre-cleared lysates were then incubated overnight at 4°C with monoclonal rabbit antibodies Anti-HA (1:250 dilution) (Clone 114-2C-7, Merck Millipore). The immuno-complexes were captured after incubation with Protein-A agarose beads (pre-saturated with BSA) during a 4 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 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 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 50 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Base calling: NextSeq Control Software 4.0.2.7, RTA2.11.3, bcl2fastq2.17 v2.17.1.14
Read Quality: FastQC (Galaxy Version 0.73+galaxy0), FASTQ Trimmer (Galaxy Version 1.1.5)
Alignment: Bowtie2 (Galaxy Version 2.4.2+galaxy0)
Peak calling: MACS2 (Galaxy Version 2.1.1.20160309.6 , No broad regions option) relative to the total input DNA samples. The q-value (false discovery rate, FDR) cut-off for called peaks was 0.05.
Motif-based sequence analysis: http://meme-suite.org/
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 DjlA-HA, DnaJ12345-HA and ClpX-HA 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 strand; column 4: Feature Type; column 5: Feature Name; column 6: Description; column 7 to 18: DjlA-HA, DnaJ12345-HA and ClpX-HA ChIP-Seq profiles (normalized Read Per Million unit, RPM)
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| Submission date |
Jul 24, 2023 |
| Last update date |
Nov 09, 2023 |
| Contact name |
Patrick H. Viollier |
| E-mail(s) |
Patrick.Viollier@unige.ch
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| Organization name |
University of Geneva, Faculty of Medicine / CMU
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| Department |
Dept. Microbiology and Molecular Medicine
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| Street address |
Rue Michel Servet 1
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| City |
Geneva 4 |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
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| Platform ID |
GPL24555 |
| Series (2) |
| GSE225489 |
Co-chaperone-mediated post-translational control of efflux pump induction underlies adaptive β-lactam resistance in Caulobacter crescentus |
| GSE238117 |
Co-chaperone-mediated post-translational control of efflux pump induction underlies adaptive β-lactam resistance in Caulobacter crescentus. [ChIP-Seq 2] |
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| Relations |
| BioSample |
SAMN36696918 |
| SRA |
SRX21141236 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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