|
| Status |
Public on Aug 21, 2021 |
| Title |
D.Melanogaster_female_whole-fly-lysate_AL-diet_ZT4_Replicate-1_Tim01 |
| Sample type |
RNA |
| |
|
| Source name |
35 female Tim01 mutant flies reared on AL-diet for seven days
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Tim01
time of collection: ZT 4 (12:00pm)
diet: Ad libitum (5% yeast-extract)
|
| Treatment protocol |
Four-day old mated female flies (Canton-S and Tim01) were sorted on either Ad libitum (5.0% yeast-extract) or Dietary Restriction (0.5% yeast-extract) and reared in a 12:12hr light-dark cycle for seven days. On the seventh day flies were collected every four hours for 20 hours (6 timepoints) starting at ZT 0 (lights on).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from whole fly lystates of approximately 35 flies (4 biological replicates per diet and time-point) with Qiagen's Rneasy Lipid Tissue Mini Kit (74804). Samples were then lysed in Qiagen's Qiazol lysis buffer with Qiagen's TissueLyser (pulverized for approximately nine minutes). Qiagen's QIAcube robot was used to futher proccess the samples utilizing a modified version of Qiagen's QIAcube protocol: RNeasy Lipid Tissue Mini-Animal Tissues-Aqueous Phase.
|
| Label |
Cy3
|
| Label protocol |
For each experimental sample, approximately 150ng of total RNA was amplified with Sigma's TransPlex Complete Whole Transcriptome Amplification Kit (WTA2). Qiagen's QIAquick PCR Purification Kit (28104) was used to purify the amplified samples. Following purification, the samples were further processed on the QIAcube according to the manufacturers' protocol: QIAquick PCR Purification-Standard. For each biological sample the NimbleGen (Roche) One-Color DNA Labeling kit (05223555011) was used to label dscDNA with Cy3 according to the manufacturers' protocol.
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| |
|
| Hybridization protocol |
Samples were hybridized to the 12-plex Gene Expression array platform according to the manufacturers' protocol. NimbleGen's Sample Tracking Controls (05223512001), Hybridization Kit reagents (05583683001) and Wash Buffers (05584507001) were used. Each 12-plex array was sealed with a H12 mixer, provided with the Gene Expression array. 4µg of each Cy3 labeled sample was loaded onto an array on the 12-plex chip, using a Gilson M100 pipette. Hybridization was completed overnight on a NimbleGen Hybridization System 4 unit, at 42℃ for 16hrs.
|
| Scan protocol |
After the 12-plex chip was washed and dried, the chip was scanned on a GenePix 4200A scanner at 300PMT, 100POW. Arrays were then quantitated using NimbleGen's NimbleScan2 software.
|
| Description |
processed data file: Tim01_AL_JTK_CYCLE.txt
This sample is from mated Tim01 mutant females reared on AL (5% yeast-extract) diet for seven days and collected at ZT 4. RNA was extracted and pooled from whole-fly lysates from approximately 35 flies. This sample is the first of four replicates.
|
| Data processing |
Arrays were normalized using LIMMA (Smyth & Speed 2003); The *Cycle data provides the processed data that has been analyzed with the JTK_CYCLE algorithm.
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| |
|
| Submission date |
Aug 20, 2021 |
| Last update date |
Aug 21, 2021 |
| Contact name |
Brian A Hodge |
| E-mail(s) |
bhodge@buckinstitute.org
|
| Phone |
7193737545
|
| Organization name |
Buck Institute
|
| Lab |
Kapahi
|
| Street address |
338 EDGEWOOD AVE, A
|
| City |
MILL VALLEY |
| State/province |
CA |
| ZIP/Postal code |
94941-2630 |
| Country |
USA |
| |
|
| Platform ID |
GPL15486 |
| Series (1) |
| GSE158286 |
Diet-specific circadian transcriptome of Canton-S and Tim01 females. |
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