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Sample GSM4105679

Query DataSets for GSM4105679

Status

Public on Apr 20, 2020

Title

Sample 9_PAO1_3

Sample type

SRA

Source name

PAO1_3

Organism

Pseudomonas aeruginosa PAO1

Characteristics

genotype/variation: wild-type
cell type: Bacterial cells grown to OD 0.5
treatment protocol: no rifampicin treatment
chip antibody: Agarose-immobilized anti-VSVG (Sigma-Aldrich, Cat.# A1970, monoclonal antibody clone P5D4)

Treatment protocol

As indicated above, a subset of the samples were treated with rifampicin (150 µg/mL) for 30 minutes prior to the ChIP protocol,

Growth protocol

Bacterial cultures were started from single colonies, grown overnight, and back-diluted in LB media

Extracted molecule

genomic DNA

Extraction protocol

DNA associated with proteins of interest were immunoprecipitated from lysates with anti-VSVG monocolonal antibody
Immunoprecipitated DNA was not size-selected during library construction
Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following manufacturer’s instructions.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

mock IP: IP with the anti-VSVG antibody on lysates from cells lacking the target epitope

Data processing

Paired ChIP-Seq reads corresponding to fragment sizes of either 200 or 400 bp (see SAMPLES section) were aligned to the PAO1 genome using bowtie2 (version 2.3.1) allowing 1 mismatch in a 28 bp seed region with no discordant or unpaired alignments.
The program samtools (version 1.2) was used to extract read 1 from each mapped read pair.
For the RsmA-V +/- rifampicin experiments (samples 1-2): Peaks were identified using QuEST (version 2.4; Valouev et al., 2008) using control data from exponential-phase wild-type cells (sample 3) with the following settings: KDE = 50, seeding fold enrichment = 1.25, extension fold enrichment = 1.5, ChIP to background fold enrichment = 2
For Hfq +/- ∆rsmAF (Samples 4 and 5) ChIP Experiment: Peaks were identified using QuEST (version 2.4; Valouev et al., 2008) using control data from log-phase wild-type cells (Sample 9) with the following settings: KDE = 45, seeding fold enrichment = 1.25, extension fold enrichment = 1.5, ChIP to background fold enrichment = 2.
For RsmA +/- ∆hfq (Samples 7 and 8) ChIP Experiment: Peaks were identified using QuEST (version 2.4; Valouev et al., 2008) using control data from log-phase wild-type cells (Sample 9) 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 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.
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 signicant (as defined above in data processing steps) enrichment of reads in the experimental condition in comparision to the mock control condition.

Submission date

Oct 02, 2019

Last update date

Apr 20, 2020

Contact name

Michael J Gebhardt

Organization name

University of Iowa

Department

Department of Microbiology and Microbiology

Lab

Gebhardt