|
| Status |
Public on Sep 01, 2014 |
| Title |
mpeg1 morphant_uninfected_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
zebrafish embryo injected with mpeg1 morpholino1, not-infected
|
| Organism |
Danio rerio |
| Characteristics |
morpholino injection at 1-cell stage: mpeg1 mo1 0.2 mM
blood island injection at 28 hpf: PBS/2%PVP
developmental stage rna isolation: 4 dpi
|
| Treatment protocol |
Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro-injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock-injected with PBS/2%PVP as a control. At 5 days post fertilization (4 days post infection) pools of 30 embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using QIAZOL 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) containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 0,7 ml of QIAzol® Reagent (Qiagen) and subsequently total RNA was extracted and on-column DNase digestion was performed with a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies).
A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina Inc., San Diego, USA). In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA-Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs (sample 25) and 3 sequencing runs (samples 2, 11-16, 24 and 26) were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments.
Sequence reads were quality trimmed using the quality_trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts (Zv9_67) using the ref_assemle_short module in the CLCbio Assembly Cell v4.0.6.
Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly_table module in the CLCbio Assembly Cell v4.0.6. Secondly, a custom script was used that sums all reads belonging to the same gene. Non-uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally, read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level.
Fold-change and differential expression significance values were calculated from gene level read counts using the DESeq package (version 1.8.3) available in Bioconductor (version 2.10).
Genome_build: Danio_rerio.Zv9.67.dna.toplevel / Danio_rerio.Zv9.67.cdna.all.fa (http://www.ensembl.org/info/data/ftp/index.html)
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample
|
| |
|
| Submission date |
Feb 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Annemarie H. Meijer |
| E-mail(s) |
a.h.meijer@biology.leidenuniv.nl
|
| Organization name |
Institute of Biology, Leiden University
|
| Department |
Animal Sciences and Health
|
| Street address |
Einsteinweg 55
|
| City |
Leiden |
| ZIP/Postal code |
2333 CC |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL14875 |
| Series (1) |
| GSE54885 |
Macrophage-Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti-Bacterial Function in Zebrafish |
|
| Relations |
| BioSample |
SAMN02640193 |
| SRA |
SRX469449 |
