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| Status |
Public on Feb 06, 2014 |
| Title |
wildtype1 |
| Sample type |
SRA |
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| Source name |
whole embryo
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| Organism |
Tribolium castaneum |
| Characteristics |
strain: San Bernardino
tissue: whole embryo
age: 10-11 h @ 32 °C
type: wild type
RNAi: dsRNA template: -
read length: 100bp
read type: single end
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| Treatment protocol |
dsRNA was generated as described in (Posnien N. et al, Cold Spring Harb Protoc. 2009 Aug;2009(8):pdb.prot5256). 400 female pupae were injected with dsRNA and crossed with wild type males. Staged egg collections of 10-11 h old embryos were used for RNA extraction.
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| Growth protocol |
Beatles were reared at 32 °C on white flour supplemented with 5% dry yeast
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Trizol (Ambion) according to the manufacturers protocol. Crude RNA extracts were DNAse digested with Turbo DNAse (Ambion) followed by Phenol/Chloroform (Ambion) extraction according to the manufacturers protocol.
Library preparation for RNA-Seq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 400 ng of total RNA . Accurate quantitation of cDNA libraries was performed by using the QuantiFluor™ dsDNA System (Promega). The size range of final cDNA libraries was determined applying the DNA 1000 chip on the Bioanalyzer 2100 from Agilent (280 bp). cDNA libraries were amplified and sequenced by using the cBot and HiSeq2000 from Illumina (SingleRead; 1x50 bp and 1x100bp).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
technical triplicate, summed up, treated as single sequencing run
Reference (fastqc): FastQC A Quality Control tool for High Throughput Sequence Data http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ by S. Andrews
Reference (bowtie2): Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359.
Reference (samtools): Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., and Subgroup, 1000 Genome Project Data Processing (2009). The Sequence Alignment/Map format and SAMtools. Bioinforma. Oxf. Engl. 25, 2078–2079.
Reference (DESeq): Anders, S., and Huber, W. (2010). Differential expression analysis for sequence count data. Genome Biol. 11, R106.
Reference (BLAST): Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.
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| Data processing |
Sequence images were transformed with Illumina software BaseCaller to bcl files, which were demultiplexed to fastq files with CASAVA v1.8.2. Quality check was done via fastqc (v. 0.10.0, Babraham Bioinformatics).
Reference transcriptome: The au2.mrna gene set was used as the mapping reference. Isoforms were removed to avoid multiple mappings. The final transcriptome contained only t1 isoforms.
Reads were mapped to the reference using bowtie2 (version 2.1.0) with the these settings: -q -D 20 -R 3 -N 1 -L 20 -i S,1,0.50
Raw count tables were obtained with samtools (version 0.1.18).
Statistical analysis was performed with DESeq (version1.12.0). Genes are considered differentially expressed if log2FoldChange >= |1| and p_adjusted<0.1
Independent filtering was applied with theta=0.25 . All other settings were default.
Gene prediction set au2: http://bioinf.uni-greifswald.de/tcas/genes/au2/
Supplementary_files_format_and_content: Unnormalized count tables are tab-delimited text files with gene ID, length and count number, e.g. sample1.counts.txt
Supplementary_files_format_and_content: Output tables of DEseq are comma-separated text files with: gene ID, base mean of normalized counts, fold change, log2 fold change, p-values, adjusted p-values
Supplementary_files_format_and_content: Annotation of results: The au2 gene set was BLASTed against the gene set from Flybase using BLAST implemented in bioperl and a custom perl script (script written by Mario Stanke). Reciprocal best BLAST hits were reported as orthologs. Hits with an E-value < 10^-5 were considered homologs. The resulting list of au2 gene IDs with their corresponding Fbpp ID was merged to the Drosophila GO annotations from Flybase (GOC Validation Date: 01/25/2013) in R.
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| Submission date |
Feb 05, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Georg Oberhofer |
| E-mail(s) |
goberho@gwdg.de
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| Phone |
+49 176 877 87615
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| Organization name |
University Göttingen
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| Department |
Developmental Biology
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| Lab |
Bucher
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| Street address |
Justus von Liebig Weg 11
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| City |
Göttingen |
| State/province |
Lower Saxony |
| ZIP/Postal code |
37077 |
| Country |
Germany |
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| Platform ID |
GPL18255 |
| Series (1) |
| GSE54706 |
Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone. |
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| Relations |
| BioSample |
SAMN02628807 |
| SRA |
SRX465154 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1322429_sample18.counts.txt.gz |
90.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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