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| Status |
Public on Apr 20, 2020 |
| Title |
Sample 5 A_Hfq_delAF_ChIP |
| Sample type |
SRA |
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| Source name |
Hfq_delAF_ChIP
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| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
genotype/variation: Hfq-VSVG {delta}rsmA {delta}rsmF
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)
input/mock used: Sample 6_PAO1_2
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| 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,
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| Growth protocol |
Bacterial cultures were started from single colonies, grown overnight, and back-diluted in LB media
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| 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| 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.
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| Submission date |
Oct 02, 2019 |
| Last update date |
Apr 21, 2020 |
| Contact name |
Michael J Gebhardt |
| Organization name |
University of Iowa
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| Department |
Department of Microbiology and Microbiology
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| Lab |
Gebhardt
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| Street address |
431 Newton Road, 200A EMRB
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
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| Platform ID |
GPL18782 |
| Series (1) |
| GSE138338 |
Widespread targeting of nascent transcripts by RsmA in Pseudomonas aeruginosa |
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| Relations |
| BioSample |
SAMN12898295 |
| SRA |
SRX6938684 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4105669_Hfq-V_delAF_AllPeaks.bed.gz |
19.0 Kb |
(ftp)(http) |
BED |
| GSM4105669_Hfq-V_del_rsmAF_A_ChIP_normalized.profile.wig.gz |
19.8 Mb |
(ftp)(http) |
WIG |
| GSM4105669_Hfq-V_del_rsmAF_A_background_normalized.profile.wig.gz |
20.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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