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| Status |
Public on Jun 09, 2020 |
| Title |
ChIP-Seq_CtrA_NA1000∆divJ∆pleC |
| Sample type |
SRA |
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| Source name |
ChIP-Seq_CtrA_Caulobacter_crescentus_NA1000∆divJ∆pleC
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| Organism |
Caulobacter vibrioides |
| Characteristics |
genotype: {delta}divJ{delta}pleC
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| Treatment protocol |
80 ml of mid-log phase cells (OD660~ 0.5-0.6) were cross-linked in 1% formaldehyde and 10 mM sodium phosphate (pH 7.6) at room temperature for 10 min and 30 min on ice thereafter. Crosslinking was stopped by addition of 125 mM glycine and incubated for 5 min on ice. Cells were washed thrice in PBS resuspended in 450 µl in TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl)
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| Growth protocol |
C. crescentus strains grown overnight at 30°C in complex media (PYE) to saturation before being diluted in fresh PYE media and grown at 30°C to mid-exponential phase (OD660~0.5-0.6).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Resuspended pellets were lysed with 2 µl of Ready-lyse lysozyme solution (Epicentre) for 5 min at RT. Protease inhibitors (Roche) was added and incubated for 10 min. Then, 550 µl of ChIP buffer (1.1% triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, plus protease inhibitors) was added to the lysate and incubated at 37 °C for 10 min before sonication (2 x 8 bursts of 30 sec on ice using a Diagenode Bioruptor) to shear DNA fragments to a length of 300 to 500 bp. Lysate was cleared by centrifugation for 10 min at 12,500 rpm at 4 °C and protein content was evaluated by measuring OD280. Then, 7.5 mg of proteins were diluted in ChIP buffer supplemented with 0.01% SDS and precleared 1 h at 4 °C with 50 µl of protein A agarose beads (BioRad) and 100 µg BSA. Two µl of polyclonal anti-CtrA antibodies were added to the supernatant before overnight incubation at 4 °C under gentle agitation. Eighty µl of BSA pre-saturated protein A agarose beads were added to the solution for 2 h at 4 °C with rotation, 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), once with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), once with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), once with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA) at 4 °C and a second wash with TE buffer at RT. The DNA-protein complexes were eluted twice in 250 µl freshly prepared elution buffer (0.1 M NaHCO3, 1% SDS). NaCl was added at a concentration of 300 mM to the combined eluates (500 µl) before overnight incubation at 65 °C to reverse the crosslink. The samples were treated with 20 µg of proteinase K in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5) for 2 h at 45 °C. DNA was extracted using Nucleospin PCR clean-up kit (Macherey-Nagel) and resuspended in 50 µl elution buffer (5 mM Tris-HCl pH 8.5). Sample libraries of immunoprecipitated chromatin and deep-sequencing were performed at the Genomicscore KULeuven (Belgium).
ChIP-Seq libraries were prepared at the Genomicscore KULeuven (Belgium) using the Illumina TruSeq DNA library according to the manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Around 2 x 10^7 single-end sequence reads (1 x 50) were first mapped on the genome of C. crescentus NA1000 (NC_011916.1) and converted to SAM using BWA and SAM tools from the sourceforge server (https://sourceforge.net/)
MACS2 algorithm was used to model the length of DNA fragment as well as the shift size.
The number of reads overlapping each genomic position was computed using custom Python scripts and the previously modeled DNA fragment and shift sizes.
A peak was defined as the genomic region where each position has more reads than the 97th percentile. The candidate peaks were annotated using custom Python scripts.
In the purpose to compare strains, the total number of reads was normalized by the ratio of the number of reads between the two strains.
Genome_build: NC_011916.1
Supplementary_files_format_and_content: The xls files report CtrA ChIP-Seq peaks in the WT and mutant strains of Caulobacter. The different columns are organized as follows: column 1: Start_Coord (coordinates for the start of the peak); column 2: End_Coord (coordinates for the end of the peak); column 3: Summit (highest number of reads of peak); column 4: Summit_Coord (coordinates for the highest number of reads of peak); column 5: Reads (number of reads in the peak); column 6: Reads Norm (number of reads / total number of alignedreads ); column 7: GeneUp (CCNA_number of gene upstream of the peak based on the summit); column 8: GeneUpName (name of gene upstream of the peak); column 9: GeneDown ( CCNA_number of gene downstream of the peak based on the summit); column 10: GeneDownName (name of gene downstream of the peak); column 11: OperonUp (Operon in which gene upstream of the peak belongs); column 12: OperonDowncolumn (Operon in which gene downstream of the peak belongs).
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| Submission date |
Jun 08, 2020 |
| Last update date |
Jun 09, 2020 |
| Contact name |
Regis Hallez |
| E-mail(s) |
[email protected]
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| Organization name |
UNamur
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| Street address |
61 Rue de Bruxelles
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| City |
Namur |
| ZIP/Postal code |
5000 |
| Country |
Belgium |
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| Platform ID |
GPL22063 |
| Series (1) |
| GSE152025 |
Regulation of bacterial cell cycle progression by redundant phosphatases |
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| Relations |
| BioSample |
SAMN15168980 |
| SRA |
SRX8498585 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4598966_ChIPSeq_CtrA_deltadivJpleC_Table.xlsx |
52.0 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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