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| Status |
Public on Jun 27, 2018 |
| Title |
wild type larval GMC; age:5h - replicate2 |
| Sample type |
SRA |
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| Source name |
larval brain
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: GMC
developmental stage: third instar larva
marker: cells express nuclear GFP marker (UAS-stinger::GFP)
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| Growth protocol |
Flies expressing UAS-dcr2; ase-GAL4, UAS-stinger::GFP;; were raised at 25C. Wandering L3 larval brains were collected and prepared for FACS-sorting. We cultured sorted type I NBs for 1.5h, 3h or 5h, where they continued to divide asymmetrically. We subjected the cell suspension containing NBs and newly formed GMCs to a second FACS sort to collect pure populations of both daughter cells.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Type I neuroblasts and GMCs of different ages were FACS sorted and total RNA was isolated using TRIzol LS reagent. Total RNA was converted to first strand (using oligo-(dT)20 primers) and second strand cDNA. Library preparation was done using Illumina's protocol with Nextera DNA Library Preparation Kit using a modified Index primer 1 (i7), which included random octamere sequences for molecular barcoding.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Bascalling performed using RTA 1.18.61.0
The unstranded reads were screened for ribosomal RNA by aligning with BWA (v0.7.12) against known rRNA sequences (RefSeq).
The rRNA subtracted reads were aligned with TopHat (v2.1.1) against the Drosophila melanogaster genome (FlyBase r6.12). Introns between 20-150000 bp are allowed which is based on FlyBase statistics. Microexon-search was enabled. Additionally, a gene model was provided as GTF (FlyBase r6.12).
Reads arising from duplication events are marked as such in the alignment (SAM/BAM files) as follows. The different tags are counted at each genomic position. Thereafter, the diversity of tags at each position is examined. First, tags are sorted descending by their count. If several tags have the same occurrence, they are further sorted alphanumerically. Reads sharing the same tag, are sorted by the average PHRED quality. Again if several reads have the same quality, they are further sorted alphanumerically. Now the tags are cycled through by their counts. Within one tag, the read with the highest average PHRED quality is the unique correct read and all subsequent reads with the same tag are marked as duplicates. Furthermore, all reads which have tags with one mismatch difference compared the pool of valid read tags are also marked as duplicates.
snRNA, rRNA, tRNA, snoRNA and pseudogenes are masked from the GTF (FlyBase r6.12) with subtractBed from bedtools (v2.26.0).
The aligned reads were counted with HTSeq (v0.6.1; intersection-nonempty).
The genes were subjected to differential expression analysis with DESeq2 (v1.12.4).
Genome_build: FlyBase r6.12
Supplementary_files_format_and_content: Excel files (xlsx) containing the differential expression analysis (standard DESeq2 output); text file of counts (HTSeq count output)
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| Submission date |
Sep 20, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Sebastian Wissel |
| Organization name |
Institute of Molecular Biotechnology (IMBA)
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| Street address |
Dr. Bohr-Gasse 3
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL17275 |
| Series (1) |
| GSE104049 |
poly-A RNA profiling of Drosophila neural stem cells (type I NBs) and GMCs of different ages reveal genes involved in cell fate stabilization |
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| Relations |
| BioSample |
SAMN07674804 |
| SRA |
SRX3200859 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2788348_GMC5h_16745_2_H7EMPADXX.count.txt.gz |
60.6 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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