|
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 |