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| Status |
Public on May 01, 2020 |
| Title |
NtrX 50 mM ChIP-seq |
| Sample type |
SRA |
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| Source name |
Bacterial culture
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| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
media: SIS medum
oxygen: 30% Oxygen
tissue: Bacterial culture
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| Growth protocol |
R. sphaeroides 2.4.1 cultures were grown in Sistrom’s minimal medium (SMM), with 4 g/L succinate. 500 ml cultures were grown in Roux bottles bubbled with 30% O2, 1% CO2, and 69% N2. 50 µM IPTG was added at inncoulation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
R. sphaeroides cultures were grown aerobically in 500 ml cultures. At OD600 = 0.3-0.4, formaldehyde and sodium phosphate were added to a final concentration of 1% and 10 mM, respectively. This mixture was incubated at 30 °C for 4 min before glycine was added to 100 mM, and the solution was placed on ice for 30 min with gentle agitation to quench excess formaldehyde. Cells were centrifuged at 3000g, washed twice with chilled phosphate-buffered saline, centrifuged, and flash-frozen at − 80 °C. About 2 × 1010 cells were suspended in 0.5 ml of 100 mM Tris (pH 8.0), 300 mM NaCl, 2% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, and sonicated eight times for 20 s with a Branson Sonifier (Branson Ultrasonics Corp., Danbury, CT) set to level 6 and 50% output using a 3-mm microtip. A mixture of micrococcal nuclease (50 U) and RNase A (0.5 μg) in 200 μM CaCl2, 1.2 mM KCl, 6 mM sucrose, and 10 μM DTT was added; the mixture was incubated for 1 h at 4 °C; and then nuclease activity was inhibited by addition of 10 mM ethylenediaminetetraacetic acid (EDTA). Cell debris was removed by centrifugation for 10 min at 12,000g, and an aliquot was removed to analyze DNA fragmentation by agarose gel eletrophoresis (desired size of ∼ 200–1000 bp with enrichment for ∼ 500-bp molecules). The resulting supernatant was incubated with gentle mixing with 20 μl of Staphylococcus aureus protein A Sepharose beads (Sigma-Aldrich, St. Louis, MO) for 3 h at 4 °C as pretreatment to remove potential nonspecific binding to the beads. After the beads had been removed by centrifugation (5 min at 3000g), one-tenth of the sample was removed and used as non-antibody-treated control. This supernatant was treated with polyclonal antibodies against the Myc epitope tag (ab9132, Abcam), and the mixture was incubated overnight at 4 °C with gentle mixing before being incubated with protein A Sepharose beads (30 μl) for 2 h at 4 °C. The beads were recovered by centrifugation; washed once (4 °C) with 250 mM LiCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; washed twice with 600 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; washed twice with 300 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; and washed twice with TE buffer (10 mM Tris pH 8.0 and 1 mM EDTA). Protein–DNA complexes were eluted from the beads by incubating at 65 °C for 30 min in 50 mM Tris (pH 8.0), 10 nM EDTA, and 1% sodium dodecyl sulfate. The beads were removed by centrifugation, and protein–DNA cross-linking was reversed by incubating the samples for 12 h at 65 °C. DNA was purified using the QIAquick PCR Purification Kit (QIAGEN, Inc., Valencia, CA).
ChIP-seq library preparation and sequencing was performed at the University of Wisconsin-Madison Biotechnology Center. Libraries for sequencing were created using the Illumina TruSeq ChIP library preparation kit (Illumina) following the standard protocol. ChIP-seq libraries were sequenced on an Illumina HiSeq 2500 2x150bp reads using the standard protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
For ChIP-seq analysis, the initial 50bp sequence tags were mapped to the R. sphaeroides 2.4.1 genome using SOAP version 2.21, allowing a maximum of 2 mismatches and no gaps. Peaks were identified using MOSAiCS at a FDR of 0.05. The MOSAiCS analysis was conducted as a two-sample analysis, with ChIP-seq data from either input DNA or ChIP conducted with anti-Myc antibody in R. sphaeroides 2.4.1 without a Myc-tagged protein used as a control. Only peaks that were called as significant using both controls (i.e., the intersect of the 2 analysis) were considered as true peaks. The analysis was conducted using cells grown with 3 and 50 μM IPTG
Genome_build: GCA_000012905.2
Supplementary_files_format_and_content: Normalized RPKM read counts for RNA-seq. WIG files for ChIP-seq
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| Submission date |
Feb 18, 2020 |
| Last update date |
May 02, 2020 |
| Contact name |
Kevin S Myers |
| E-mail(s) |
kmyers2@wisc.edu
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| Organization name |
University of Wisconsin - Madison
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| Department |
Great Lakes Bioenergy Research Center
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| Street address |
5127 WEI, 1552 University Ave
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53726 |
| Country |
USA |
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| Platform ID |
GPL18842 |
| Series (1) |
| GSE145442 |
The NtrYX two-component system regulates the bacterial cell envelope |
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| Relations |
| BioSample |
SAMN14124441 |
| SRA |
SRX7738715 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4318428_NtrX_50uM_ChIP-seq.wig.gz |
9.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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