|
| Status |
Public on Aug 25, 2017 |
| Title |
trr_Mutant_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Drosophila heads
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: head
developmental stage: adult (0-5 days old)
genotype: Elav-Gal4 / + ; dicer2 / + ; P{TRiP.HMS01019}attP2 / +
extraction protocol: Total RNA was extracted from 50 µl aliquots of frozen fly heads using the RNAeasy lipid tissue mini kit (Qiagen). For trr knockdown, two biological replicates were used, for G9a mutants, three biological replicates were used for each condition. mRNA was purified using the Oligotex kit (Qiagen) and cDNA was synthesized using the SuperScript III First Strand synthesis kit (Thermo Fisher) using random hexamers as primers. Second strand cDNA was synthesized using E. Coli polymerase and T4 ligase (New England Biolabs Inc. (NEB)). Remaining RNA was removed using 2 units RNaseH (NEB) before the cDNA was purified using the MinElute PCR purification kit (Qiagen). DNA end repair was performed followed by ligation of Illumina sequencing adaptors and size selection for 300 bp by 2% E-gel (Invitrogen). Fragments were linearly amplified (15 PCR cycles), as validated by quantitative real-time PCR (qPCR), and sample quality was assessed using the Agilent Bioanalyser 2100.
|
| Treatment protocol |
Wild type, UAS/Gal4 mediated knock down of Trr and G9a mutant Drosophila melanogaster heads
|
| Growth protocol |
Flies were cultured according to standard methods at 25 degrees with a 12 hour light dark cycle
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
cDNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~300 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
none
|
| Data processing |
Quality control: fastqc and Htseq_qa.py
RNA-seq: Reads were mapped to the Drosophila genome (BDGP R5/dm3 using the Burrows Wheeler Aligner (BWA, version 0.6.1) with standard settings allowing 1 mismatch. Only the uniquely mapped reads were used for further analysis and total alignment efficiency was between 63% and 79% for G9a samples and between 79% and 81% for trr samples. Total read count data was generated by the python script HTSeq-count (http://www-huber.embl.de/HTSeq/doc/overview.html) with gene annotations extracted from the file Drosophila_melanogaster.BDGP5.75.gtf, available at http://www ensembl.org. In all samples, over 96% of aligned reads, mapped unambiguously to exons. The unambiguously mapped reads, ranging from 18 to 25 million reads for G9a samples and from 26 to 28 million reads for trr samples, were used for further analysis of differential gene expression by DeSeq2. Hierarchical clustering based on euclidean distances with pearson correlation using the normalized expression values and variance stabilizing tranformation, revealing a high degree of similarity between biological replicates.
genome build: BDGP R5/dm3
processed data files format and content: text file with raw counts.
|
| |
|
| Submission date |
Nov 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tom Koemans |
| E-mail(s) |
tom.koemans@radboudumc.nl
|
| Organization name |
Radboudumc
|
| Department |
Human genetics
|
| Lab |
van Bokhoven/ Schenck
|
| Street address |
Geert Grooteplein 10
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11203 |
| Series (1) |
| GSE89459 |
Genome wide binding of trr (ChIP-seq) and expression analysis (RNA-seq) of trr- and G9a mutant fly heads |
|
| Relations |
| BioSample |
SAMN05968769 |
| SRA |
SRX2321794 |
