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| Status |
Public on Jan 18, 2019 |
| Title |
NPF_1_RNAseq |
| Sample type |
SRA |
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| Source name |
neuropeptide F neuron nuclei
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| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: UAS-unc84-2XGFP; NPF-GAL4
neuronal population: neuropeptide F
replicate: 1
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| Growth protocol |
Flies were raised at 25°C in a 12-h light/12-h dark cycle in 60% relative humidity and maintaned on cornmeal, yeast, molasses, and agar medium. Approximately 300 heads were collected from 2-3 day old flies.
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Heads were separated by vigorous vertexing followed by separation over dry-ice cooled sieves. 9ml of homogenization buffer (20mM β-Glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40 supplemented with RNAase inhibitor,10mg/ml t-RNA, 50mg/ml ultrapure BSA, 0.5mM Spermidine, 0.15mM Spermine and 140ul of carboxyl Dynabeads -270 Invitrogene: 14305D) was added to each sample. The heads were minced on ice by a series of mechanical grinding steps followed by filtering the homogenate using a 10um Partek filter assembly (Partek: 0400422314). After removing the carboxyl-coated Dynabeads using a magnet, the homogenate was filtered using a 1um pluriSelect filter (pluriSelect: 435000103). The liquid phase was carefully placed on a 40% optiprep cushion layer and centrifuged in a 4oC centrifuge for 30min at ~2300Xg. The homogenate/Optiprep interface was incubated with anti-GFP antibody (Invitrogen: G10362) and protein G Dynabeads (Invitrogen: 100-03D) for 40 minutes at 4oC . Beads were then washed once in NUN buffer (20mM β-Glycerophosphate pH7, 300mM NaCl, 1M Urea, 0.5% NP40, 2mM EDTA, 0.5mM Spermidine, 0.15mM Spermine, 1mM DTT, 1X Complete protease inhibitor, 0.075mg/ml Yeast torula RNA, 0.05Units/ul Superasin). Bead-bound nuclei were separated using a magnet stand, and RNA was extracted using the Picopure Kit (Invitrogen KIT0204).
The NuGEN RNAseq v2 (7102-32) kit was used to prepare cDNA from the INTACT purified RNA, followed by library preparation using the SPIA - NuGEN Encore Rapid DR prep kit. Samples were sequenced on an Illumina HiSeq using single-end 60 base pair reads.
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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 |
Low quality bases were trimmed from RNA-seq reads using Trim Galore and mapped to the dm6 using STAR (v2.4.2) --twopassMode.
For gene expression analysis: featureCounts from the Subread package (v1.4.5) was used to determine read counts overlapping exons in the BDGP6.88 transcriptome from ensembl. DESeq2 was used to calculate normalized counts and gene expression differences from these counts.
For RNA editing analysis: Picard tools were used to mark duplicates and samtools mpileup function was used to determine base calls at known editing sites from non-duplicate, uniquely mapped reads. Editing levels were calculated as (G reads)/(A+G reads) at each site.
Genome_build: dm6
Supplementary_files_format_and_content: For gene expression analysis: read counts for transcripts from featureCounts. Tab-delimited files are formatted as: gene id, chr, start, end, strand, length, counts.
Supplementary_files_format_and_content: For RNA editing analysis: A and G counts and editing levels at known editing sites. Tab-delimited files are formatted as: chr, position, a counts, g counts, editing level.
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| Submission date |
Apr 25, 2018 |
| Last update date |
Jan 20, 2019 |
| Contact name |
Galit Shohat-Ophir |
| E-mail(s) |
[email protected]
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| Organization name |
Bar Ilan University
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| Street address |
The Nanotechnology Building 206, Room C-663
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| City |
Ramat Gan |
| ZIP/Postal code |
5290002 |
| Country |
Israel |
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| Platform ID |
GPL17275 |
| Series (2) |
| GSE113661 |
Measuring A-to-I RNA editing and gene expression signatures of neuronal populations within the Drosophila brain |
| GSE113663 |
A-to-I RNA editing signatures of neuronal populations within the Drosophila brain |
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| Relations |
| BioSample |
SAMN08981569 |
| SRA |
SRX3994100 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3110778_npf_1_RNAseq_editing.txt.gz |
46.8 Kb |
(ftp)(http) |
TXT |
| GSM3110778_npf_1_counts.txt.gz |
686.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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