|
| Status |
Public on Apr 25, 2022 |
| Title |
GcsR_C |
| Sample type |
SRA |
| |
|
| Source name |
gcsR-vsvG
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
genotype: gcsR-vsvG
growth phase: Bacterial cells grown to OD 2.0
chip antibody: Agarose-immobilized anti-VSVG antibody (Sigma-Aldrich, catalog number A1970, monoclonal antibody clone P5D4)
|
| Treatment protocol |
At the time of harvest, 80 mL of culture was treated with formaldehyde (1% final concentration) for 30 minutes with gentle agitation to form cross-links between DNA and protein. Following this, Glycine was added to a final concentration of 250mM and incubated for 15 minutes at room temperature with gentle agitation to quench the crosslinking reaction. The cells were washed thrice with PBS and stored at -80C until further processing.
|
| Growth protocol |
Bacterial cultures were started from single colonies, grown overnight to saturation, and back-diluted in LB media to an OD600~0.01 and subsequently cultured to an OD600 of 2.0 before harvesting.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA associated with proteins of interest were immunoprecipitated from lysates with anti-VSVG monocolonal antibody
Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following manufacturer’s instructions. Size selection of DNA fragments was not used.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Sample 2 C
Immunoprecipitated DNA was not size-selected during library construction
GcsR-V_AllPeaks.bed
|
| Data processing |
Paired ChIP-Seq reads corresponding to fragment sizes of 200 bp (see SAMPLES section) were aligned to the PAO1 genome using bowtie2 (version 2.3.5.1) allowing 1 mismatch in a 28 bp seed region with no discordant or unpaired alignments.
The program samtools (version 1.9) was used to extract read 1 from each mapped read pair.
For GcsR and FleQ experiments (samples 2, 5): Peaks were identified using QuEST (version 2.4; Valouev et al., 2008) using control data from wild-type PAO1 cells (sample 1) with the following settings: KDE = 45, seeding fold enrichment = 1.25, extension fold enrichment = 1.5, ChIP to background fold enrichment = 2.0, positive region size = 300.
For GacA ChIP experiments (sample 4): Peaks were identified using QuEST (version 2.4; Valouev et al., 2008) using control data from a parental strains lacking the retS gene (PAO1 ∆retS, sample 3). The following settings were used for peak identification during QuEST analysis: KDE = 45, seeding fold enrichment = 1.25, extension fold enrichment = 1.5, ChIP to background fold enrichment = 2.0, positive region size = 300.
Bed files for each experimental replicate were created by QuEST and the final processed bed files were generated by identifying the largest overlapping peak region which statisfy the following conditions: (1) peak regions must have a positive peak shift, (2) peak regions must have a positive strand correlation, (3) peak regions must have a q-value of less than 0.01, and (4) peak regions must be shared by two or more experimental replicates.
Assembly: NC_002516.2
Supplementary files format and content: wig files were generated using QuEST; Scores represent normalized smoothed read density
Supplementary files format and content: bed intervals reflect regions with signicant (as defined above in data processing steps) enrichment of reads in the experimental condition in comparision to the mock control condition.
|
| |
|
| Submission date |
Apr 05, 2022 |
| Last update date |
Apr 26, 2022 |
| Contact name |
Joseph Mougous |
| Organization name |
University of Washington /HHMI
|
| Street address |
1959 NE Pacific St Box 357710
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL23999 |
| Series (1) |
| GSE200262 |
Genome-wide protein–DNA interaction site mapping using a double strand DNA-specific cytosine deaminase |
|
| Relations |
| BioSample |
SAMN27361425 |
| SRA |
SRX14753743 |
