|
| Status |
Public on Apr 27, 2016 |
| Title |
Dmau_TAM16_repB |
| Sample type |
SRA |
| |
|
| Source name |
eye-antennal imaginal disc
|
| Organism |
Drosophila mauritiana |
| Characteristics |
strain: TAM16
species: Drosophila mauritiana
Sex: female
developmental stage: late L3 larva
tissue: eye-antennal imaginal disc
|
| Growth protocol |
flies were raised at 25ºC and 12h:12h dark/light cycle in density‐controlled conditions (30 freshly hatched LI larvae per vial).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Female LIII larvae were dissected and eye-antennal imaginal discs were stored in RNALater at 120 h after egg laying. 40-50 discs were dissected per sample. Total RNA was isolated using the Trizol and the samples were DNAse I treated in order to remove DNA contamination.
Library preparation for RNA-Seq was performed using the TruSeq RNA Sample Preparation Kit starting from 500 ng of total RNA. Accurate quantitation of cDNA libraries was performed by using the QuantiFluor™dsDNA System . 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 cBot and HiSeq 2000 (Illumina):single-end reads were generated for D. mauritiana (replicates A, B and C) and for D. melanogaster samples (1x50 bp) and paired-end reads were generated for D. mauritiana (replicates D, E and F) and for D. simulans samples (2x100 bp).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Counts_Length_Published_DmauDmel.txt
Counts_Length_Direct_DmauDmel.txt
Counts_Length_Reciprocal_DmauDmel.txt
|
| Data processing |
Illumina Casava1.8.2 software used for basecalling.
100 bp PE RNA-seq reads (from D. mauritiana replicates D, E and F and from D. simulans A, B and C) were split into two 50 bp reads each. Left and right reads were merged into a single fastq file to be equivalent to single-end reads.
Bowtie2 with parameters –very-sensitive-local –N 1 was used to map the RNA-seq reads to the respective references: D. melanogaster reads were mapped to the published CDS set (Flybase, r5.55, only longest transcript) and to our novel reciprocally re-annotated transcript set. D. mauritiana and D. simulans reads were mapped to the respective published transcript sets, to the directly re-annotated transcript sets and to the reciprocally re-annotated transcript sets.
The number of reads mapping to each transcript were summarized using samtools v0.1.19. Transcript length was also obtained using samtools.
Genome_build: This study was perfomed with the aim of showing the bias introduced to differential expression analysis by using non-comparable species-specific mapping references. Therefore the RNA-seq reads were mapped to different transcriptomic references already published or generated in this study. D. melanogaster reads were mapped to the published CDS set (Flybase, r5.55, only longest transcript) and to our novel reciprocally re-annotated transcript set. D. mauritiana and D. simulans reads were mapped to the respective published transcript sets (Nolte, V. et al. 2013 and Hu, T.T. et al. 2013, respectively), to the directly re-annotated transcript sets and to the reciprocally re-annotated transcript sets. All re-annotated references are available upon request.
Supplementary_files_format_and_content: Files are in tab-delimited text format. The row names are the FlyBase ID of all orthologs present in the annotated transcript set for each pair of species (D. mauritiana and D. melanogaster or D. mauritiana and D. simulans). The first three columns contain the raw counts of mapped reads to the D. mauritiana references (replicates A, B and C for the comparison to D. melanogaster and replicates D, E and F for the comparison to D. simulans). Columns four to six contain the raw counts of mapped reads to D. melanogaster or D. simulans references. Column seven contains the length (in bp) of the transcript in the D. mauritiana reference and column eight contains the length (in bp) of the transcript in the D. melanogaster or D. simulans reference.
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| |
|
| Submission date |
Dec 22, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Nico Posnien |
| E-mail(s) |
nico.posnien@googlemail.com
|
| Organization name |
University of Göttingen
|
| Department |
Department of Developmental Biology
|
| Street address |
Justus-von-Liebig-Weg 11
|
| City |
Göttingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21269 |
| Series (1) |
| GSE76252 |
A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species |
|
| Relations |
| BioSample |
SAMN04361925 |
| SRA |
SRX1496466 |
