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| Status |
Public on May 31, 2023 |
| Title |
MGAS10870-CovS-T284A - output - 5 - biol rep 3 |
| Sample type |
SRA |
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| Source name |
bacteria
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| Organism |
Streptococcus pyogenes |
| Characteristics |
cell type: bacteria
strain: 10870-CovS-T284A
genotype: hyperphosphoyrlated CovR
chip antibody: anti-CovR-ND
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Three biological replicates of strains MGAS10870, M3-CovS-T284A, and 10870-ΔcovR (control), respectively, were chromatin immunoprecipitated using polyclonal antibody directed against the N-terminal part of CovR. Briefly, GAS strains were grown in 40 ml THY medium to mid-exponential phase (OD~0.45), proteins were cross-linked to DNA with 1% formaldehyde, cells harvested by centrifugation, and flash-frozen and stored at -80° C. The fixed cell pellets were resuspended in 1ml ice cold lysis buffer, and the lysates were sonicated for 15 cycles (30s on/30s off) at 4° C in 1.5 ml Bioruptor tubes (Diagenode) in a Diagenode Bioruptor Plus machine set at high power, to shear DNA to fragments of 200 and 400 bp length. The cleared supernatant was collected to use for chromatin-immunoprecipitation (950μl) or input DNA (50μl), respectively. CovR-bound DNA fragments were immunoprecipitated over night at 4°C using Dynabeads protein G (Invitrogen) pre-coated with anti-CovRND antibody. After several washing steps, the complex was eluted with 50μl elution buffer to obtain the ChIP DNA (output) samples. Proteins and RNA in both input and ChIP samples were degraded using RNaseA and proteinase K, crosslinking was reversed by incubation at 65° C overnight, and the DNA was purified using SPRI beads (AMPure XP, Beckman Coulter) on a magnetic stand. The DNA concentration was determined on a Qubit machine 4.0 (Invitrogen) following the Qubit manual for high-sensitive DNA, and DNA fragment size distribution was assessed using Agilent D1000 Screen Tape on an Agilent 2200 TapeStation system. ChIP sequencing was performed in the Advanced Technology Genomics Core (ATGC) Facility at MD Anderson Cancer Center] with Illumina compatible indexed libraries prepared from 2-10ng of sheared ChIP or input DNA. Equimolar quantities of the indexed libraries were multiplexed with 8 libraries per pool and sequenced on an Illumina NextSeq500 sequencer using the high output 75nt single read flow cell format.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequencing reads (~30-35M reads per replicate/input sample) were quality filtered, trimmed, and mapped to the reference genome MGAS10870 using CLC Genomics Workbench (v 21, Qiagen). Peaks representing potential CovR binding were identified using the Transcription Factor ChIP-seq module of CLC Genomics Workbench. Peak shape scores and RPKL values were plotted against each other and peaks manually inspected to identify a reliable threshold for statistically significant enrichment of DNA. Peaks with a peak shape score >30 and RPKL >500 in at least two of the samples were called as statistically significant. A gene was associated with an enriched DNA region if the peak center was within 200 bps of the promoter or the open reading frame of the gene.
Supplementary files format and content: Each worksheet contains ChIP-seq data for that sample
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| Submission date |
Apr 28, 2023 |
| Last update date |
May 31, 2023 |
| Contact name |
William Charles Shropshire |
| E-mail(s) |
wcshropshire@mdanderson.org
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| Phone |
8307082542
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| Organization name |
The University of Texas MD Anderson Cancer Center
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| Street address |
1901 East Road
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77054 |
| Country |
USA |
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| Platform ID |
GPL28678 |
| Series (1) |
| GSE230870 |
Hyperphosphorylation of the Group A Streptococcal Control of Virulence Regulator Increases Promoter Occupancy Specifically at Virulence Factor Encoding Genes |
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| Relations |
| BioSample |
SAMN34424675 |
| SRA |
SRX20140677 |
| 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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