|
| Status |
Public on Feb 08, 2018 |
| Title |
T5_27x_17 |
| Sample type |
SRA |
| |
|
| Source name |
PA20 cultures
|
| Organism |
Escherichia coli |
| Characteristics |
incubation time: 5 hr
ug/ml smx / ug/ml tm: 540/108 (27x)
|
| Growth protocol |
PA20 cultures for RNA-seq were grown on T-agar with and without added Sulfamethoxazole- Trimethoprim at designated levels for the indicated times and temperatures. Working stocks were tested and maintained using LB (Miller formulation) or LB agar. Plasmid pSE380 derivatives were induced by IPTG.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA samples were isolated using RNAzol RT (Molecular Research Center) by following manufature's instructions. Contaminating DNA was removed from RNA samples by DNase I digestion using the TURBO DNA-free kit (Ambion).
RNA libraries were prepared for sequencing using standard Illumina protocols; rRNAs were removed by Ribozero (gram negative), libraries were prepared using TruSeq protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
RNA-seq data were mapped to the E. coli O157:H7 Sakai genome using the Subjunc aligner program from the Subread package (v1.4.6) (http://bioinf.wehi.edu.au/subread/).
The alignment Bam files were compared against the gene annotation general feature format (GFF) file, and raw counts for each gene were generated using the featureCounts tool from Subread. The raw counts data of the expressed genes was normalized for RNA composition using the trimmed mean of M values (TMM) method (http://www.ncbi.nlm.nih.gov/pubmed/20196867) from the Empirical analysis of Digital Gene Expression Data in R (EdgeR) package (https://bioconductor.org/packages/release/bioc/html/edgeR.html), then transformed to log2CPM (counts per million) values using the voom method (http://www.ncbi.nlm.nih.gov/pubmed/24485249) from the R LIMMA package (https://bioconductor.org/packages/release/bioc/html/limma.html).
Next, a linear model was built for each comparison using the R LIMMA package and statistics for differential expression analysis were computed. To filter for differential expression, two fold or three-fold change with a FDR ≤0.05 were used as the threshold.
Functional annotation was done using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.7, NIAID, NIH (http://david.abcc.ncifcrf.gov).
Supplementary_files_format_and_content: comma-delimited text files include RPKM values for each Sample
|
| |
|
| Submission date |
Feb 07, 2018 |
| Last update date |
Feb 08, 2018 |
| Contact name |
Gaylen C Ulrich |
| E-mail(s) |
George.Paoli@ars.usda.gov
|
| Organization name |
USDA/ARS/Eastern Regional Research Center
|
| Street address |
600 East Mermaid Lane
|
| City |
Wyndmoor |
| State/province |
PA |
| ZIP/Postal code |
19038 |
| Country |
USA |
| |
|
| Platform ID |
GPL18133 |
| Series (1) |
| GSE110255 |
Sulfamethoxazole – Trimethoprim represses csgD but 2 maintains virulence genes at 30°C in a clinical 3 Escherichia coli O157:H7 isolate |
|
| Relations |
| BioSample |
SAMN04040767 |
| SRA |
SRX2345228 |
