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| Status |
Public on Jan 01, 2014 |
| Title |
Pph1448A(pBAD::hrpL)_1 |
| Sample type |
SRA |
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| Source name |
bacterial culture
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| Organism |
Pseudomonas syringae |
| Characteristics |
pathovar: phaseolicala
strain: 1448A
plasmid: pBAD::hrpLPph1448A
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| Growth protocol |
Pseudomonas strains were grown overnight at 28 ˚C, in KB media supplemented with appropriate antibiotics, then sub-cultured in fresh media at OD600= 0.2, and grown until OD600= 0.4-0.5. Bacteria were washed twice with 10 mM MgCl2 and resuspended in minimal medium (Huynh et al., 1989) supplemented 0.1% peptone. Bactria were then inoculated in supplemented minimal media at OD600= 0.2, and incubated shaking for 1 hour at 28 ˚C. Expression of hrpL was induced by addition of 200 mM L-arabinose. Aliquots of cell culture were taken 1, 3, 5 hours post-induction and treated with RNAprotect reagent (Qiagen).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was performed by using the RNeasy minikit (Qiagen). Isolated total RNA was treated with TURBO DNase (Ambion). Total RNA of each time point was pooled at 1/3 ratio. 10 μg of pooled RNA was depleted of 16S and 23S ribosomal RNA using RiboMinusTM (Invitrogen). Double stranded cDNA were prepared from ~ 1 μg ribosomal depleted RNA, using random primers (Invitrogen).
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Biological replicate #1
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| Data processing |
The PtoDC3000, Pph1448A, Por1_6, Pjap, and Pla107 reads produced using a Illumina GA II. The PsyB728A reads produced using a Illumina HiSeq system.
A GFF file was created for each genome by BLASTing transcripts to the genomic sequences and recoding gene coordinates for optimal hits. This was done using an in-house script. All genomes can be accessed and downloaded from NCBI. Transcriptomes were constructed by NCBI’s gene prediction pipeline for PtoDC3000, PsyB728A, and Pph1448A. Por1_6, Pla107, Pjap transcriptomes were constructed by JGI’s Integrated Microbial Genomes – Expert Review gene prediction pipeline.
GENEcounter was used to map RNA-seq reads from each strain to the matching genomic sequence. Used GENEcounter default parameters.
GENEcounter was used to count the number of reads mapped to each gene for each genome and detect differential expression between HrpL and EV samples within each strain.
A slight modification in GENEcounter allowing for random seeding allowed us to bootstrap the normalization. Bootstrap values were calculated for 100 random normalizations and differential expression analyses. Median count values and bootstrapped differential expression occurrences are recorded in the final processed data txt files.
Genome_build: The genomes can be accessed using the following NCBI BIoProject IDs. PtoDC3000 - PRJNA57967; PtoDC3000_draft - in-house; Pph1448A - PRJNA58099; PsyB728A - PRJNA57931; Por1_6 - PRJNA55447; Pjap - PRJNA33209; Pla107 - PRJNA33205.
Supplementary_files_format_and_content: tab-delimited text files include normalized count values for each gene in each sample
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| Submission date |
May 14, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Emma Johnson |
| E-mail(s) |
[email protected]
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| Organization name |
University of North Carolina - Chapel Hill
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| Department |
Biology
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| Lab |
Dangl Lab
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| Street address |
250 Bell Tower Dr
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| City |
Chapel Hill |
| State/province |
North Carolina |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platform ID |
GPL17159 |
| Series (1) |
| GSE46930 |
Diversity of HrpL-regulons across Pseudomonas syringae isolates |
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| Relations |
| BioSample |
SAMN02144254 |
| SRA |
SRX278028 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1141332_Pph_2HrpL.txt.gz |
23.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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