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Sample GSM4693841 Query DataSets for GSM4693841
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

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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