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| Status |
Public on Aug 06, 2016 |
| Title |
M5G_control |
| Sample type |
SRA |
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| Source name |
mixed population of Caulobacter crescentus
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| Organism |
Caulobacter vibrioides NA1000 |
| Characteristics |
phob genotype: phoB (wt)
psts genotype: pstS (wt)
antibody: anti-FLAG M2 (Sigma-Aldrich,F1804)
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| Treatment protocol |
Mid-exponential phase cultures were cross-linked in 10mM sodium phosphate and 1% formaldehyde.
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| Growth protocol |
Cultures were grown to mid-exponential phase at 30 degrees C in rich medium (PYE).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linked DNA was extracted as in Fioravanti et al., 2013 (PMID: 23737758) with modifications: mid-exponential phase cultures were cross-linked in 10 mM sodium phosphate (pH 7.6) and 1% formaldehyde at room temperature for 10 minutes. Reactions were quenched with 0.1 M glycine at room temperature for 5 minutes, and on ice for 15 minutes. Cells were washed 3X in phosphate buffered saline (PBS) and lysed with Ready-Lyse lysozyme solution (Epicentre, Madison, WI) according to manufacturer instructions. Lysates were diluted 1:1 in ChIP buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl) plus Roche Protease Inhibitor Tablets (Roche) and incubated at 37 °C for 10 minutes. Lysates were sonicated (Branson Sonicator) on ice for 6 bursts of 10 seconds each at 15% amplitude, and then cleared by centrifugation at 14,000 rpm for 5 minutes at 4 °C. Cleared supernatants were normalized by protein content in 1 mL of ChIP buffer + 0.01% SDS, and pre-cleared with 50 μL of Protein-A DynaBeads (Invitrogen) (pre-blocked with 100 μg Ultra Pure BSA in ChIP buffer + 0.01% SDS) by 1 hour rotation at 4 °C. 10% of each supernatant was removed and used as total chromatin input sample. The remaining supernatant was incubated with a 1:1000 dilution of anti-M2 antibody overnight at 4 °C. Each sample was then incubated with 50 μL of pre-blocked Protein-A DynaBeads for 6 hours at 4 °C with rotation. DynaBeads were washed consecutively at 4 °C for 15 minutes with 1 mL of the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)), and twice with TE buffer (10 mM Tris-HCl (pH 8.1), 1 mM EDTA). Complexes were eluted twice by incubation with 250 μL freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) at 30 °C for 15 minutes. Cross-links were reversed by addition of 300 mM NaCl and 2 μL of 0.5 mg/mL RNase A and overnight incubation at 65 °C. Samples were treated with 5 μL of Proteinase K (20 mg/mL, NEB) in 40 mM EDTA and 40 mM Tris-HCl (pH 6.8) for 2 hours at 45 °C. DNA was extracted using a PCR purification kit (Qiagen) and resuspended in 80 μL of water. Libraries were prepared using the SPRI-works system and sequenced on an Illumina MiSeq (MIT BioMicroCenter).
Libraries were prepared using the SPRI-works system
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Description |
Cultures were grown to mid-exponential phase at 30 degrees C in rich medium (PYE).
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| Data processing |
Reads were mapped to the Caulobacter crescentus NA1000 reference genome using BWA (Li and Durbin, 2010 (PMID: 20080505)).
860,000 uniquely mapping reads were analyzed for each sample.
Read extension and pileup was performed using MACS software analysis package (Zhang et al., 2008, PMID:18798982).
Data were analyzed using custom Python scripts and peaks were called by comparing local maxima in the treat sample to the control sample with a p-value < 10^-5
Genome_build: NC_011916.1
Supplementary_files_format_and_content: The processed wig files contain the output of MACS software analysis, with 10 bp genomic bins containing the number of reads.
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| Submission date |
Aug 07, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Laub |
| Organization name |
MIT
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| Street address |
31 Ames St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL20784 |
| Series (2) |
| GSE71860 |
Identification of the PhoB regulon and role of PhoU in the phosphate-starvation response of Caulobacter crescentus (ChIP-seq) |
| GSE71961 |
Identification of the PhoB regulon and role of PhoU in the phosphate-starvation response of Caulobacter crescentus |
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| Relations |
| BioSample |
SAMN03981705 |
| SRA |
SRX1142417 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1847007_M5G_control.wig.gz |
1.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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