|
| Status |
Public on Jan 05, 2015 |
| Title |
USA300(EX)SNM |
| Sample type |
SRA |
| |
|
| Source name |
Reference Strain
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain: USA300
media: SNM
harvest phase: Exponential
sequence events: 2
specific host: none
|
| Treatment protocol |
Stabilization of the RNA in the samples was performed by incubating with RNAprotect for 30 mins before proceeding with the RNA isolation.
|
| Growth protocol |
Two S. aureus strains (USA300 and T032) were grown in rich (BHI) and minimun media (SNM) and harvested at different phases (mid-log and stationary). The in vivo sample was taken from the anterior nares of a human nose known to be an S. aureus carrier.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen's Rneasy Mini Kit following manufacturer's instructions, the where the samples were treated by beat beating in a solution of RLT Buffer (Qiagen) and 1% of β-mercaptoethanol.
Enriched mRNA fractions were amplified using the MessageAMP II-Bacteria RNA amplification according to manufacturer's instructions. Amplified samples were converted to ds-cDNA using SuperScript Double-Stranded cDNA Synthesis Kit and purified using GeneClean Turbo for PCR Kit (MP Biomed).
Paired-End DNA Sample Prep Kit (Illumina, San Diego, USA) according to manufacturer's instructions
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Base calls for the GAIIx were done using the GA II x CASAVA-1.8.1 pipeline and for the MISeq was performed using MiSeq RTA 1.14.23 pipeline
Filtering and trimming with the following parameters: Q ≥ 15, Seq. length ≥ 80nt, Homopolymers ≤ 8nt and N's = 0.
Two sequencing events were done for all in vitro S. aureus samples. After quality filtering, libraries were merged into one file and collapsed into representative reads
From the In vivo sample (Metatranscriptome) human reads were removed using BLAT against a human genome repository downloaded from NCBI ftp site. Sequences with >70% identity were removed
Ribosomal sequences were removed using HMMER (V3.0) using as a database models build with alignments of the Ribosomal fractions (5S,16S, and 23S) and BLASTN against a database of Ribosomal proteins of S. aureus genomes
Reads were assigned as belonging to S. aureus via blastn using a database of 25 concatenated genomes
Remaining reads were blasted (RPS-BLASTN) against a reference database of Position Scoring Matrices (PSM) build from a comparison of 25 Genomes of S. aureus
Read counts were down-sampled by a random resampling using an in-house script written in perl
A rank-ordering (from highest to lowest expression) of all genes in each sample was performed
Supplementary_files_format_and_content: text file with abundance (by counting the number of transcripts) for each gene (groups of genes, OG) within each condition
|
| |
|
| Submission date |
Mar 27, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Diego Chaves-Moreno |
| Organization name |
Helmholtz Centrum for Infection Research
|
| Department |
Department of Medical Microbiology (MMIK)
|
| Lab |
Research Group Microbial Interactions and Processes (MINP)
|
| Street address |
inhoffenstrasse 7
|
| City |
Braunschweig |
| ZIP/Postal code |
38124 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16057 |
| Series (1) |
| GSE56294 |
Comparative genomics of Staphylococcus aureus and the application of a “pan-genome” for assigning RNA-Seq transcript reads from divergent strains and in vivo human nasal samples |
|
| Relations |
| BioSample |
SAMN02709626 |
| SRA |
SRX502158 |
