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| Status |
Public on Jun 01, 2015 |
| Title |
rpoA_empty2 |
| Sample type |
SRA |
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| Source name |
planktonic cells
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| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: UCBPP-PA14
genotype: delta-sutA carrying pMQ72
growth phase: late stationary
medium: minimal medium plus 40mM sodium pyruvate, 20mM arabinose and 100 µg/ml gentamicin
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| Treatment protocol |
Cultures were fixed by incubation with 1% formaldehyde for 15 min, then quenched with 125 mM glycine for 10 min, pelleted, and frozen at -80⁰C.
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| Growth protocol |
Starter cultures of each of the three strains were grown overnight in LB medium, then diluted 1:1000 into 50 ml minimal medium (14.15 mM KH2PO4, 35.85 mM K2HPO4, 42.8 mM NaCl, 9.3 mM NH4Cl, 1 mM MgSO4, 7.2 μM FeCl2 and trace elements (Widdel et al., 1983)) plus 40 mM sodium pyruvate, 20 mM arabinose, and 100 µg/ml gentamicin. Cultures were grown at 37 degrees C with shaking for 24 hours, reaching an OD of approximately 1.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Pellets were lysed by treatment with 1 mg/ml lysozyme and sonicated with a microtip sonicator for 4 minutes, alternating 30 seconds on and 30 seconds off to shear DNA. Immunoprecipitation with an anti-HA antibody pre-conjugated to agarose beads or with an anti-RpoA antibody followed by protein A/G-conjugated agarose beads was carried out as descibed by Gilbert et al (Molecular microbiology 73, 1072-1085). Extracted, RNAse-treated DNA was purified using QIAquick columns and used for library construction.
Libraries for ChIP Seq were constructed from the fragmented genomic DNA samples using the NEBNext ChIP Library Prep Master Mix Set for Illumina, according to the 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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| Description |
rpoA ChIP in ∆sutA strain, replicate 2
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| Data processing |
Image analysis, base calling, de-multiplexing, and conversion of sequencing to fastq format were carried out by HiSeq Control Software (HCS) versions 2.0.12 and 2.2.38 and Real Time Analysis (RTA) versions 1.17.21.3 and 1.18.61 on the Illumina HiSeq 2500 machine.
Reads were trimmed and filtered based on their quality scores using the Trimmomatic package v 0.30 (Bolger et al, Bioinformatics published online April1 2014). Leading and trailing quality cutoffs were set to 27, and a cutoff of 20 was set for a sliding window of 4 bases. Reads shorter than 35 bp after trimming were discarded.
Mapping trimmed reads to the Pseudomonas aeruginosa genome was carried out using Bowtie version 1.0.0 (Langmead et al., Genome biology 10, R25), with the following arguments: -n 2 --best -S. The .sam files generated by bowtie were converted to .bam files, sorted, and indexed using samtools version 0.1.19 (Li et al., Bioinformatics 25, 2078-2079).
Reads per feature were calculated using the easyRNASeq package version 1.4.2, part of the Bioconductor project for R (Delhomme et al., Bioinformatics 28, 2532-2533). Three different feature sets were used: genes, 100bp regions, and transcription units. See supplementary files for summaries
RPKM for genes, 100 bp regions, and transcription units were calculated by normalizing to reads mapped from each sample and size of each feature. Aggregated ratios and FDR values were calculated using the Degust web interface for the edgeR package (Robinson et al., Bioinformatics 26, 139-140).
Genome_build: gi|116048575|ref|NC_008463.1|
Supplementary_files_format_and_content: For each sample, a processed data file containing the counts per 100 bp in .wig format is provided. Additionally, supplementary files are provided that give the genomic loci, aggregated ratios of HASutA vs empty vector control ChIP signal, and rpkm values for each sample, for each gene in the PA14 genome; the aggregated ratios and rpkm values for each sample, for each 100 bp region; and the genomic loci and rpkm values for each transcriptional unit.
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| Submission date |
Feb 23, 2015 |
| Last update date |
Apr 16, 2020 |
| Contact name |
Dianne Newman |
| E-mail(s) |
dkn@caltech.edu
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| Organization name |
Caltech
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| Street address |
1200 E California Blvd, MC 147-75
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| City |
Pasadena |
| State/province |
CA |
| ZIP/Postal code |
91125 |
| Country |
USA |
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| Platform ID |
GPL18287 |
| Series (2) |
| GSE66179 |
Chromatin Immunoprecipitation of HA-tagged PA14_69770 and RpoA in Pseudomonas aeruginosa UCBPP-PA14 |
| GSE66181 |
SutA is a Transcription Elongation Factor Expressed During Slow Growth in *Pseudomonas aeruginosa* |
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| Relations |
| BioSample |
SAMN03365902 |
| SRA |
SRX885666 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1616440_rpoA_empty2.wig.gz |
88.6 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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