|
| Status |
Public on Nov 25, 2014 |
| Title |
P. chlororaphis_scv_AB_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
SCV mutant cells
|
| Organism |
Pseudomonas chlororaphis subsp. aureofaciens 30-84 |
| Characteristics |
genotype: SCV mutant
strain: 30-84
|
| Growth protocol |
Biological replicates of every strain were started from single colonies located on two separate plates containing AB + 2% CAA and then transferred to 1.5 ml AB + 2% CAA broth. All cultures were grown at 28˚C to an approximate OD600 = 1.8.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Transfer 700 μl lysate into RNeasy Mini column place in a 2 ml collection tube and centrifuge for 30 s at highest speed. Add 350 μl Buffer RW1 to the column and centrifuge for 30 s. Discard flow-through and reuse the collection tube. Add 10 μl DNase stock solution to 70 μ l RDD Buffer. Add the DNase I solution into the column and incubate at RT for 15 min. Add 350 μl Buffer RW1 to the column and wait for 5 min, centrifuge for 30 s. Discard the flow-through and collection tubes. Place the RNeasy Mini spin column in a new 2 ml collection tube. Add 500 μl Buffer RPE to the column. Centrifuge for 30 s. Place the column in a new 1.5 ml tube, and centrifuge for 1 min to eliminate any residue ethanol. Place the column in a new collection tube. Add 50 μl RNase free water, centrifuge for 1 min. Store RNA samples at -20°C
Strand-specific cDNA libraries were constructed using Illumina TruSeq RNA Sample Preparation Kits.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
strain: Pseudomonas chlororaphis 30-84 genotype: mutant, growing condition: AB minimal medium, Stage: late logarithm phase
Strand-specific cDNA libraries were constructed using the SOLiD Total RNA-Seq kit according to the manufacturer’s instructions with the following modifications: 1) For RNA fragmentation, 300 ng of rRNA-depleted RNA were incubated at 95˚C for 5 min. Bioanalyzer traces were used to confirm that the largest proportion of RNA fragments were around 200 bp. 2) Bioanalyzer traces following reverse transcription were used to confirm that hybrization and ligation of RNA adapters and subsequent reverse transcription of the RNA to cDNA was successful. 3) Amplification of cDNA was carried out following reverse transcription, but preceding the size-selection step. Samples were barcoded by replacing the SOLiD 3’ primer with the appropriate barcoding primer as directed in the SOLiD instructions. Size selection of DNA amplicons ranging from 200-300 bp was performed using an E-Gel iBase system (SYBR Safe 2% SizeSelect gel) (Life Technologies, Carlsbad, CA). This step was repeated to ensure that adapter-adapter dimers were fully eliminated before sequencing. Paired-end sequencing was carried out by the University of Texas Genomic Sequencing and Analysis Facility (UTGSAF: http://www.ti3d.utexas.edu/facilities/gene-expression) on a Life Technologies SOLiD 5500xl sequencing system. Targeted sequencing depth was six-million paired-end reads per sample.
|
| Data processing |
Paired-end sequencing was carried out by the Otogenetic company. Targeted sequencing depth was ten-million paired-end reads per sample.
Based on gene annotation information, the pipeline produced genome alignment results as a compressed binary version of the Sequence Alignment Map (BAM files).
Based on gene annotation information, the pipeline produced genome alignment results as a compressed binary version of the Sequence Alignment Map (BAM files).
Mapped reads were visualized using BamView in Artemis 13.2.0 (Rutherford et al. 2000).
The number of reads per kb of transcript per million mapped reads (RPKM) was used to normalize the raw data (Mortazavi et al., 2008), and mean RPKM values were determined for both the wild-type and mutant samples.
Genome_build: GenBank Accession #: PRJNA67533
Supplementary_files_format_and_content: Excel file containing the RPKM values for each gene
|
| |
|
| Submission date |
Sep 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
DONGPING WANG |
| E-mail(s) |
[email protected]
|
| Phone |
2177225029
|
| Organization name |
OilDri Corporation of America
|
| Department |
Amlan
|
| Street address |
777 Forest Edge Dr
|
| City |
Vernon Hills |
| State/province |
IL |
| ZIP/Postal code |
60061 |
| Country |
USA |
| |
|
| Platform ID |
GPL16805 |
| Series (1) |
| GSE51293 |
Genes differentially expressed in Pseudomonas chlororaphis 30-84 SCV |
|
| Relations |
| BioSample |
SAMN02370140 |
| SRA |
SRX361896 |
