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| Status |
Public on Aug 27, 2013 |
| Title |
am12 |
| Sample type |
SRA |
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| Source name |
anterior midgut
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Oregon R
Stage: 6-14 day old adult female
subregion: a1
replicate: 3
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| Growth protocol |
Fertilized adult females 4-14 days of age were employed to avoid the final steps of gut maturation that take place in young adults, as well as age-induced decline. Flies were kept at a controlled density in fresh vials, at 25°C and provided with a uniform level of nutrition before and during the study period. Under these conditions, the cellular structure of the midgut was stable and reproducible as assessed by cell counts along its length.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
We identified GAL4 lines with regional expression, then dissected and sectioned gut regions based on the presence or absence of GFP under a fluorescent dissecting microscope. Gut regions were isolated, no more than 5 at a time, and moved to iced tripure (Roche/Boehringer Mannheim cat: 11667157001) to avoid RNA degradation (which occurred unless guts were processed within 30 min of isolation (or 10-15 minutes in the case of the copper region samples). After 25 or 50 gut regions were collected in 200μl tripure, they were homogenized. 600μl of fresh tripure was added, mixed, and allowed to stand at room temperature for 5-10 min. After adding 180μl chloroform, samples were vortexed 2 x 45 secs, and incubated at room temperature for 10 minutes. Samples were then centrifuged for 15 min at 12,000 rpm at 4°C, and the aqueous layer was moved to a fresh tube. RNA was precipitated by adding 400μl of isopropanol, vortexing for 15 seconds, and incubating at room temperature for 15 min, and centrifuging at 12,000 rpm for 15 min at 4°C. After washing the pellet with 75% EtOH, the RNA was dried in air dry for 5 minutes, resuspended in 50μl nuclease free H2O, and stored at -80°C.
cDNA libraries were constructed from poly(A)-selected RNA using Illumina TruSeq RNA Library Prep Kit v2
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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 |
basecalling: CASAVA v1.8.2
reads aligned to genome using Bowtie2 v 2.0.6
identified reads hitting transcripts using TopHat v 2.0.7 with -G --no novel juncs and Refseq dm3-iGenomes.gtf
calculated transcript fpkm values and gene fpkm values using Cufflinks version 2.02 -G with dm3-iGenomes.gtf
Genome_build: Refseq annotation file:dm3 and dm3-iGenomes.gtf
Supplementary_files_format_and_content: Cufflinks output file: genes.fpkm_tracking:
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| Submission date |
Jun 10, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Allan C Spradling |
| E-mail(s) |
spradling@ciwemb.edu
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| Phone |
410 246-3015
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| Organization name |
Carnegie Institution/HHMI
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| Department |
Embryology
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| Street address |
3520 San Martin Dr.
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21218 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE47780 |
Drosophila midgut regional gene expression |
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| Relations |
| Reanalyzed by |
GSM3283772 |
| BioSample |
SAMN02194152 |
| SRA |
SRX298291 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1159612_am12genes.txt.gz |
435.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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