|
| Status |
Public on Dec 11, 2014 |
| Title |
M. marinum E11 5DPI [IL-13-08_Anita_39_CGATGT_L001_R1_001] |
| Sample type |
SRA |
| |
|
| Source name |
embryo 5 days post fertilization
|
| Organism |
Danio rerio |
| Characteristics |
infection: M. marinum E11 mCherry
|
| Treatment protocol |
Zebrafish embryos were micro-injected into the yolk (64 cell stage) with M. marinum E11 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), or Non-injected as a control. At 5 days post injection 50 embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.
|
| 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°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent (Invitrogen) and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Wildtype; injected with M. marinum into the yolk at 2 hpf; RNA isolated at 5 days post injection. Biological replica 2
|
| Data processing |
GeneTiles was used for quantification and visualization of the RNAseq data
Bowtie2 is used to align the reads in the fastq file to the genome (obtained from Ensemble). Bowtie2 generates SAM files that contain the reads together with the location on the genome.
Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome, resulting in a sorted BAM file.
The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome, i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file.
Using the available annotation from Ensemble we can search the BAM file for reads within a gene (using exon starting positions and lengths). This is done with a python script. The output of this script is a tab separated file (tsv) containing the read counts per gene.
We used DESeq, an R-script to perform statistical analysis. The DESeq script is used to normalise the reads per gene, based on (divided by) the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P-value, by calculating the dispersion per gene using DESeq. The size factors as well as the P-values are stored in ‘tsv’ files.
Using a script, all tsv files are combined into tsv files , e.g. per experiment, chromosome, per filtered results of most significant reads or highest ratio between measurement and control.
Genome_build: Zv9_toplevel
Supplementary_files_format_and_content: txt file containing read counts per gene
|
| |
|
| Submission date |
May 19, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Wouter Veneman |
| E-mail(s) |
w.j.veneman@biology.leidenuniv.nl
|
| Organization name |
Leiden University
|
| Department |
Institute Biology Leiden
|
| Lab |
Molecular Cell Biology
|
| Street address |
Einsteinweg 55
|
| City |
Leiden |
| State/province |
Zuid-Holland |
| ZIP/Postal code |
2333CC |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL14875 |
| Series (1) |
| GSE57792 |
Gene expression profiling of zebrafish embryos at 5 days post fertilization |
|
| Relations |
| BioSample |
SAMN02786805 |
| SRA |
SRX546379 |
