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| Status |
Public on Jun 16, 2018 |
| Title |
Saureus_RsaA_HybridTrapSeq |
| Sample type |
SRA |
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| Source name |
HG003
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| Organism |
Staphylococcus aureus |
| Characteristics |
strain: HG003
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| Extracted molecule |
total RNA |
| Extraction protocol |
Hybrid-trap-seq experiments were run in parallel with 4 sRNAs used as bait (RsaE compared to three control sRNAs ie RNAIII, RsaA and RsaH). The background noise due to nonspecific RNA binding was filtered out from RsaE dataset by a differential analysis using the three other sRNA datasets as control. Synthetic RNAs were generated with the T7 MEGAshortscript kit (Ambion) according to manufacturer’s instructions and using as DNA matrix sRNA gene PCR-amplified. Synthetic RNAs were 3’-end biotinylated as previously described (Jestin et al., 1997, DOI:10.1093/emboj/16.10.2945) using biotinamidocaproyl hydrazide (Sigma-Aldrich, B3770). After the biotinylation reaction, full length RNAs were purified on 5 % urea PAGE. Running Hybrid-trap-seq requires a pool of total RNA extracts to be used as prey. Total RNA samples were extracted in 16 different biological conditions (previously described in GEO and corresponding to submission GSE104971). Twenty µg of each of the 16 total RNA extracts were pooled to obtain the combined RNA extracts sample used during the procedure. For each Hybrid-trap-seq experiment, MasterBeads pre-coated with streptavidin (Ademtech, Pessac-France) were equilibrated in binding buffer (20 mM Tris-HCl, 0.5 M NaCl pH 8) and incubated 10 min at 20°C with 100 pmoles of biotinylated sRNA. Unbound sRNAs were removed by magnetic separation, and then sRNA-bound streptavidin beads were washed twice with binding buffer. 50 µg of pooled total RNA sample were mixed with the sRNA-bound beads. After 15 min at 45°C, followed by 15 min at room temperature, the unbound RNAs were removed by magnetic separation. The RNA-bound beads were washed twice with wash buffer (7 mM Tris-HCl pH 8, NaCl 0.17 M) before elution in RNase-free water.
Strand-specific RNA-seq libraries were generated with the TruSeq RNA-Seq Library Preparation Kit (Illumina), according to the manufacturer's instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
RNA enriched in Hybrid-Trap-seq data with RsaA sRNA as bait.
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| Data processing |
FastQC (v0.10.1) quality control.
Mapping performed on Refseq CP000253.1 by Bowtie software with parameters -p 12 -S -m 1 -q .
The number of reads mapping to each predicted CDS retrieved from Genbank file CP000253.1 and 44 bona fide sRNAs (from Liu F. et al 2018, doi: 10.3389/fmicb.2018.00228) was calculated by HTSeq-count (Anders, Pyl et al. 2015).
Differential expression analysis (normalisation and fold change calculation) was performed with DESeq software. Fold changes were computed from normalized read number of condition A (defined by the three control sRNAs datasets) to normalized read number of condition B (defined by the RsaE dataset). Genes were considered as putative RsaE targets if with a fold change >= 10.
Genome_build: CP000253.1
Supplementary_files_format_and_content: The processed data file (putative_RsaE-targets_revised_20180425.csv) compiles read counts for the 4 RNA-seq samples and DEseq output for RsaE sample versus control samples.
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| Submission date |
Oct 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Tatiana Rochat |
| E-mail(s) |
[email protected]
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| Organization name |
INRA
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| Lab |
Virologie et Immunologie Moléculaires
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| Street address |
Domaine de Vilvert
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| City |
Jouy-en-Josas |
| ZIP/Postal code |
78350 |
| Country |
France |
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| Platform ID |
GPL16057 |
| Series (2) |
| GSE106327 |
The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus [Hybrid-Trap-seq] |
| GSE106457 |
The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus |
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| Relations |
| BioSample |
SAMN07947884 |
| SRA |
SRX3343296 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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