|
| Status |
Public on Oct 26, 2010 |
| Title |
salm_inf_2 (s_2) |
| Sample type |
SRA |
| |
|
| Source name |
pooled whole zebrafish embryos injected with Salmonella typhimurium
|
| Organism |
Danio rerio |
| Characteristics |
tissue: whole organism
age: 35 hours post fertilization
|
| Treatment protocol |
Zebrafish embryos were infected with Salmonella typhimurium (strain SL1027) by microinjection of DsRED-labeled bacteria into the caudal vein close to the urogenital opening after the onset of blood circulation (27 hours post fertilization). Samples were collected at 8 hours post injection and immediately quenched in liquid nitrogen and stored at -80 ˚C prior to RNA extraction. For the duration of bacteria injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma). The total RNA of three biological samples was pooled using equal amounts of RNA
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5-30°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tag library preparation was performed using the DGE: Tag Profiling for NlaIII Sample Prep kit from Illumina according to the manufacturer’s instructions. In brief, 1 g of total RNA was used for mRNA capture using magnetic oligo(dT)beads. First- and second-strand cDNA was synthesized and bead-bound cDNA was subsequently digested with NlaIII. Fragments other than the 3 cDNAfragments attached to oligo(dT) beadswerewashed away and a GEX NlaIII adapter was ligated to the free 5 -end of the digested bead-bound cDNA fragments. The GEX NlaIII adapter contains a restriction site for MmeI which cuts 17–18 bp downstream from the NlaIII site, thereby releasing 21–22 bp tags starting with the NlaIII recognition sequence, CATG. A second adapter (GEX adapter 2) was ligated at the site of MmeI cleavage, and the adapter-ligated cDNA tags were enriched using PCR-primers that anneal to the adaptorends. The resulting 85 bp fragments were purified from a 6% acrylamide gel. Purity and yield were checked by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
The total RNA of three biological samples was pooled using equal amounts of RNA. The library was sequenced using 3 pmol of cDNA.
|
| Data processing |
Image analysis, base calling, extraction of 17 bp tags and tag counting were performed using the Illumina pipeline. Tag counts from duplicate libraries were merged in silico.
|
| |
|
| Submission date |
Jun 21, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Oliver Stockhammer |
| E-mail(s) |
o.w.stockhammer@amc.uva.nl
|
| Organization name |
AMC
|
| Department |
Medical Microbiology
|
| Street address |
Meibergdreef 15
|
| City |
Amsterdam |
| State/province |
NH |
| ZIP/Postal code |
1105 AZ |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL9319 |
| Series (1) |
| GSE22472 |
Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection |
|
| Relations |
| SRA |
SRX022404 |
| BioSample |
SAMN00016452 |
