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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 30, 2013 |
| Title |
AH_FruP14/440_1 |
| Sample type |
SRA |
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| Source name |
Adult Head Tissue
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: unknown
genotype: Df(3R)P14/Df(3R)fru4-40
Sex: Male
developmental stage: adult
tissue: head
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| Growth protocol |
Flies were raised on standard cornmeal food medium at 25˚C on a 12 hour light and 12 hour dark cycle.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol® Reagent (Invitrogen), and RNA was precipitated by addition of 250 µL 100% isopropanol and 250 µL 1.2 M NaCitrate, 0.8 M NaCl in DEPC-treated water. The Zymo Research RNA Clean & Concentrator™-25 In-Column DNase Digestion protocol and 10 units Ambion® TURBO™ DNase was used on ~25 µg total RNA. Poly(A)+ transcripts were subsequently isolated from total RNA using Ambion® MicroPoly(A)Purist™ Kit.
One hundred ng mRNA was chemically fragmented to a range of approximately 200-500 base pairs using the Ambion® RNA Fragmentation Reagent, and the reaction was cleaned using Zymo Research RNA Clean & Concentrator™-5. First strand cDNA was synthesized using SuperScript® II Reverse Transcriptase (Invitrogen™) and a combination of 3 µg random hexamers and 0.15 µg oligo(dT)20 primers. The second strand of the cDNA was synthesized using DNA polymerase I (Invitrogen™), RNase H (New England Biolabs® Inc.), dNTPs and second strand buffer (Invitrogen™). This reaction and all subsequent reactions were cleaned using Zymo Research DNA Clean & Concentrator™-5 kit. Double stranded cDNA templates were blunt ended using End-It™ Repair Kit (Epicentre®). A-overhangs were added to both ends with Klenow fragment (3'→5' exo-minus) (New England Biolabs® Inc.) and Illumina sequencing adapters were then ligated to both ends of the cDNA templates using Fast-Link™ DNA Ligation Kit (Epicentre®). cDNA templates were amplified by performing PCR for 18 cycles, which extended the adapter and incorporated a different six base pair index into each sample. The product was then isolated by gel purification of 250-550 base pair fragments.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Basecalls performed using CASAVA version 1.3
Barcode, primer, and adapter sequences were trimmed using custom perl scripts..
Reads were aligned to the D. melanogaster genome FB5.30 (FlyBase v5.30) using Bowtie (v0.12.8; --tryhard, --best, --strata, -m1).
Unaligned reads were 3’ end quality trimmed and homopolymers (5+) were removed. Quality trimmed reads were mapped again using bowtie.
Unaligned reads were aligned to junctions estimated by Tophat.
Any remaining unaligned reads were mapped to FB5.30 genome using LAST (v193; -l 20, -Q3).
Using FlyBase annotions, overlapping exons regardless of strand were combined into the maximum exonic region.
Expression was then quantified for each exonic region using Reads Per Kilobase Per Million Mapped Reads (RPKM).
Genome_build: Flybase 5.30
Supplementary_files_format_and_content: Are in the CSV format. With the following columns: exonic region id, sample id, number of mapped reads, number of reads in the exonic region, coverage in exonic region (number of reads in exonic region / read length), exonic region length, average per nucleotide coverage, RPKM.
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| Submission date |
Aug 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Michelle N Arbeitman |
| E-mail(s) |
michelle.arbeitman@med.fsu.edu
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| Organization name |
Florida State University
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| Department |
Medicine Biomedical Sciences
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| Street address |
1115 W. Call Street
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| City |
Tallahassee |
| State/province |
Florida |
| ZIP/Postal code |
32306 |
| Country |
USA |
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| Platform ID |
GPL11203 |
| Series (1) |
| GSE50515 |
Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains |
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| Relations |
| Reanalyzed by |
GSM3284681 |
| BioSample |
SAMN02360993 |
| SRA |
SRX357300 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1220621_coverage_on_fusions_2011-05-03_1_GATCAG.csv.gz |
1.4 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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