|
| Status |
Public on Dec 23, 2020 |
| Title |
AML85.1.1 C |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells grown to OD 0.35
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
genotype: PAO1 <delta>mvaU <delta>mvaT <delta>pilY1
growth phase: mid-logarithmic phase culture
chip antibody: Agarose-immobilized anti-VSVG antibody (Sigma-Aldrich, catalog number A1970, monoclonal antibody clone P5D4)
|
| Treatment protocol |
Samples were crosslinked with formaldehyde at 1% final concentration for 30 minutes, then the reaction was quenched with addition of glycine to a final concentration of 250 mM. Cell pellets were washed 3 times with PBS and frozen at -80°C for further processing/extraction.
|
| 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 cultures were grown with aeration at 250 rpm at 37°C to a final OD600 ~0.35. For RpoD-V ChIP samples, 15 mL of culture was also reserved and processed for RNA-seq.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA associated with proteins of interest were immunoprecipitated from lysates with anti-VSVG monoclonal 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 HiSeq 2500 |
| |
|
| Description |
Sample 6 C
PAO1 ΔmvaU ΔmvaT ΔpilY1
Immunoprecipitated DNA was not size-selected during library construction.
RpoD-V_delUT_AllPeaks.bed
RpoD-V_delUT_C_background_normalized.profile.wig.gz
RpoD-V_delUT_C_normalized.profile.wig.gz
|
| Data processing |
Paired ChIP-Seq reads corresponding to fragment sizes of 200 bp 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 MvaT-V and MvaU-V ChIP experiments (samples 2 and 3, respectively): 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.
For PAO1 ∆pilY1 RpoD-V ChIP experiments (sample 5): Peaks were identified using QuEST (version 2.4; Valouev et al., 2008) using control data from ∆pilY1-parental cells (sample 4) with the following settings: KDE = 30, seeding fold enrichment = 1.25, extension fold enrichment = 1.5, ChIP to background fold enrichment = 2.
For PAO1 ∆pilY1 ∆mvaU ∆mvaT RpoD-V ChIP experiments (sample 7): Peaks were identified using QuEST (version 2.4; Valouev et al., 2008) using control data from PAO1 ∆pilY ∆mvaU ∆mvaT-parental cells (sample 6) with the following settings: KDE = 30, seeding fold enrichment = 1.25, extension fold enrichment = 1.5, ChIP to background fold enrichment = 2.
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 satisfy 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.
Genome_build: 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 significant (as defined above in data processing steps) enrichment of reads in the experimental condition in comparison to the mock control condition.
|
| |
|
| Submission date |
Jul 23, 2020 |
| Last update date |
Dec 23, 2020 |
| Contact name |
Andrew M Lippa |
| Organization name |
Boston Children's Hospital
|
| Department |
Pediatrics, Division of Infectious Diseases
|
| Lab |
Dove Lab
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL18782 |
| Series (2) |
| GSE155024 |
H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription [ChIP-seq] |
| GSE155025 |
H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription |
|
| Relations |
| BioSample |
SAMN15636537 |
| SRA |
SRX8816657 |
