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| Status |
Public on Feb 03, 2021 |
| Title |
ChIP-Seq_TrcR_WTRm1021_S.meliloti_PYE |
| Sample type |
SRA |
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| Source name |
Sinorhizobium meliloti 2011
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| Organism |
Sinorhizobium meliloti 2011 |
| Characteristics |
strain: Rm2011
genotype: Wild type
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| Treatment protocol |
Cells were cross-linked in 10 mM sodium phosphate (pH 7.6) and 1% formaldehyde at room temperature for 10 minutes and on ice for 30 minutes thereafter and washed three times in phosphate-buffered saline (PBS).
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| Growth protocol |
Mid-log phase C. crescentus cells were cultured in PYE and S. meliloti cells cultured in LB supplemented with CaCl2 2.5 mM and MgSO4 2.5 mM or with an extra 10 minutes treatment with antibiotics (Rifampicin 30 μg/ml for C. crescentus and 100 μg/ml for S. meliloti ; Nalidixic Acid 20 μg/ml).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in a Ready-Lyse lysozyme solution (Epicentre Technologies) according to manufacturer’s instructions and lysates were sonicated (Bioruptor® Pico) in an ice-water bath for 15 cycles 30 seconds ON and 30 seconds OFF, to shear DNA fragments to an average length of 0.3-0.5 kbp and cleared by centrifugation at 14,000 g for 2 minutes 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-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl plus protease inhibitors (Roche, Switzerland) and pre-cleared with 80 μL of protein-A (rabbit antibodies) or –G (mouse antibodies) agarose (Roche) and 100 μg BSA. Ten percent of the supernatant was removed and used as total chromatin input DNA as described before (Delaby et al., 2019).Two microliters of polyclonal antibodies to TrcR, or of a mix of RNA polymerase from a monoclonal antibody sampler kit (ratio 1:1:1:1, Biolegend), or of monoclonal antibodies to σ70 (BioLegend, Mouse IgG2b) were added to the remains of the supernatant, incubated overnight at 4 °C with 80 μL of protein-A or -G agarose beads pre-saturated with BSA, washed once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl) and LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)), and twice with TE buffer (10 mM Tris-HCl (pH 8.1) and 1 mM EDTA). The protein DNA complexes were eluted in 500 μL freshly prepared elution buffer (1% SDS and 0.1 M NaHCO3), supplemented with NaCl to a final concentration of 300 mM and incubated overnight at 65 °C to reverse the crosslinks. The samples were 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 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’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 |
Base calling: HiSeq Control Software 2.2.58, RTA 1.18.64.0, CASAVA-1.8.2
Alignment: Map with Bowtie for illumina (Galaxy Version 1.2.0), 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 v1.46 (Braham Bionformatics Institute)
Genome_build: Caulobacter crescentus : NC_011916.1, Sinorhizobium meliloti chromosome : NC_020528.1, Sinorhizobium meliloti pSymA: NC_020527.1 and Sinorhizobium meliloti pSymB: NC_020560.1
Supplementary_files_format_and_content: The xls file reporting TrcR, RpoD and RNAP ChIP-Seq peaks 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). Common peaks between TrcR, RNAP, RpoD and CdnL are highlighted.
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| Submission date |
Apr 14, 2020 |
| Last update date |
Feb 03, 2021 |
| Contact name |
Patrick Viollier |
| Organization name |
University of Geneva UNIGE
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| Department |
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 |
GPL28391 |
| Series (2) |
| GSE148652 |
The DUF1013 protein TrcR tracks with RNA polymerase to control bacterial cell cycle and protection against antibiotics (ChIP-seq dataset) |
| GSE148654 |
The DUF1013 protein TrcR tracks with RNA polymerase to control bacterial cell cycle and protection against antibiotics |
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| Relations |
| BioSample |
SAMN14596588 |
| SRA |
SRX8114664 |
| 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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