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| Status |
Public on Aug 14, 2014 |
| Title |
med27 RNAi central brain pupal neuroblasts 1 (Tech. Rep. 1) |
| Sample type |
SRA |
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| Source name |
pupal brain
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: Neuroblasts
Stage: 0-2hr after pupa formation
marker: cells express membrane-bound GFP marker (UAS-CD8::GFP)
treatment: med27 RNAi
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| Growth protocol |
Flies expressing UAS-CD8::GFP in background containing empty Landing site VIE-260B (VDRC TID:60100) or UAS-CD8::GFP and med27 RNAi (VDRC TID:106703) under inscuteable-Gal4 were flipped on fresh food, left to lay for 24 h, flipped into fresh vials and then kept at 29oC degree for five or 6 days respectively, before dissection of late 3rd instar larval or 0-2hrs pupal brains.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Central brain neuroblasts (large cells, high GFP expression) were FACS sorted from wild type (TID:60100) or med27 RNAi and RNA was isolated using Trizol reagent. Total RNA(~1µg) was enriched for polyA mRNA, fragmented and first strand and second strand (dUTP, instead dTTP was used) was generated. Library preparation was done using a modified protocol from Illumina with NEBNext DNA sample Prep Reagent kits (NEB), and UGDase treatment was done to confer strand specificity.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls performed using RTA v1.13.48.0
The strand specific paired-end reads were screened for ribosomal RNA by aligning with BWA (v0.6.1) against known rRNA sequences (RefSeq). Non matching reads were considerd downstream.
The rRNA subtracted paired-end reads were aligned with TopHat (v1.4.1) against the Drosophila melanogaster genome (FlyBase r5.44) and a maxiumum of 6 missmatches. Introns between 20-150000 bp are allowed which is based on FlyBase statistics. Maximum multihits was set to 1 and InDels as well as Microexon-search was enabled. Additionally, a gene model was provided as GTF (FlyBase r5.44).
Aligned reads in valid pairs are subjected to FPKM estimation with Cufflinks (v1.3.0). In this step bias detection and correction was performed. Furthermore, only those fragments compatible with FlyBase annotation (r5.44) were allowed and counted towards the number of mapped hits used in the FPKM denominator. snRNA, rRNA, tRNA, snoRNA and pseudogenes are masked.
The aligned reads were counted with HTSeq. Counts for technical replicates are summed up. The polyA containing transcripts were subjected to differential expression analysis with DESeq (v1.8.3). DESeq parameters were chosen to match the DESeq publication (Anders S, Huber W., 2010).
Genome_build: FlyBase dmel_r5.44
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample (Standard output cufflinks); tab-delimited text file with differentially expressed genes for the whole analysis (standard output DESeq).
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| Submission date |
Dec 12, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Thomas Rainer Burkard |
| E-mail(s) |
[email protected]
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| Organization name |
IMBA
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| Street address |
Dr. Bohrgasse 7
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE53265 |
Ecdysone and Mediator trigger a metabolic switch uncoupling cell cycle from cell growth to end proliferation in Drosophila neural stem cells |
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| Relations |
| Reanalyzed by |
GSM3285005 |
| BioSample |
SAMN02442688 |
| SRA |
SRX390977 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1288758_cufflinks.PBNbMed27IR_12311_1_D1E5AACXX.txt.gz |
495.7 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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