|
| Status |
Public on Jun 10, 2013 |
| Title |
mRNA.6009.2h.Cefto |
| Sample type |
SRA |
| |
|
| Source name |
JKD6009
|
| Organism |
Staphylococcus aureus |
| Characteristics |
antibiotic: Ceftobiprole
growth time: 2 hours
rna preparation: mRNA
|
| Treatment protocol |
Antibiotics exposure was for 10 minutes at 0.5 x MIC.
|
| Growth protocol |
S. aureus were grown in Mueller Hinton broth for 2 or 6 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Rneasy Kit (Qiagen). RNAs were then treated with the MICROBExpress kit (AMBION) to limit the contamination by rRNAs, following manufacturer’s recommendations. At each step, RNA quality was controlled by Bioanalyzer (Agilent) using the RNA Nano Chips, and quantity was assessed using the ND-8000 (NanoDrop Technologies. Extracted RNA was prepared to enrich for mRNA or sRNA as follows. For mRNA preparations, initial fragmentation using Zinc was followed by treatment with first Antartic Phosphatase and then with PNK enzyme. Single-stranded ligation of the 3’ Illumina v1.5 adapter was followed by single-stranded ligation of 5’ bar-coded adapter. Reverse transcription and PCR amplification was performed to generate a cDNA colonies template library, and gel purification with selection of fragments of 20-300 nts was performed. Quality control of the DNA colony template library was performed by cloning an aliquot into a TOPO plasmid and sequencing 8 clones. The same process was used for sRNA preparation, without the Zinc fragmentation, and using a size selection of 20-200 nt.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Read clipping using nesoni clip tool. Nesoni url: http://vicbioinformatics.com/software.nesoni.shtml
Read alignment using SHRiMP 2.2.2. SHRiMP url: http://compbio.cs.toronto.edu/shrimp/
Alignment selection using nesoni filter tool. Only top scoring alignments were kept. Where there were multiple equal best alignments, one was chosen at random.
Counting of alignments to annotated CDS sequences and sRNA sequences, using nesoni count tool. Alignments were required to be on the correct strand.
Normalization factors to render expression levels comparable between samples were calculated using BioConductor package edgeR's implementation of the TMM normalization method.
Genome_build: CP002120.1
Supplementary_files_format_and_content: The *-count.csv files contain counts of the numbers of reads aligning to each feature. The accompanying *-counts-norm.txt files give normalization factors for each sample.
|
| |
|
| Submission date |
Sep 13, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Ben Howden |
| E-mail(s) |
bhowden@unimelb.edu.au
|
| Organization name |
Austin Health
|
| Department |
Infectious Diseases Department
|
| Lab |
Austin Centre for Infection Research
|
| Street address |
Austin Health
|
| City |
Heidelberg |
| State/province |
VIC |
| ZIP/Postal code |
3084 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16057 |
| Series (1) |
| GSE40864 |
Investigating the sRNA and mRNA transcriptional response to antibiotics in methicillin-resistant Staphylococcus aureus using Illumina RNAseq |
|
| Relations |
| SRA |
SRX186578 |
| BioSample |
SAMN01173994 |
