|
| Status |
Public on Apr 15, 2018 |
| Title |
Sample1_exp3_chip1_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
1st instar male larva; 51-61 hours post laying; 245-520 individuals per replicate
|
| Organism |
Nasonia vitripennis |
| Characteristics |
Sex: male
developmental stage: 1st instar larva
tissue: whole body
strain: AsymCX
|
| Treatment protocol |
Samples were prepared at 25º C as follows: Approximately 200 N. vitrirpennis (AsymCX) virgins were collected as black pupa. When eclosed, half of them were provided with males and were allowed to mate overnight, and the other half were kept as virgins. Virgin setting result in all-male progeny whereas mated females produced ~89.5% female progeny under the design used here. Females were provided 15- 20 Sarchophaga bullata hosts in groups of 20 females for 24 hrs to induce production of eggs. The hosts were then removed and females were left overnight (~18 hrs). To collect embryos, individual females were given access to a host at one end (to restrict the oviposition site) and allowed to lay eggs for 6-10 hrs before being removed. Embryos were then harvested immediately (early embryos), 18 hrs later (late embryos), or 51 hrs later (1st instar larvae). All embryos and larvae were collected in an RNase-free environment. The host was cracked open and the "cap" removed to expose embryos. Dissecting needles were used to gently scrape embryos from the surface of the host and transfer them into a 1.5 ml tube pre-chilled on dry ice. Samples were stored at -80º C. If at anytime the host was punctured or embryos were exposed to host hemolymph, they were discarded. Estimates of the number of embryos per replicate (three per life stage/sex) were recorded; early embryos ranged from 300-900, late embryo 140-500, 1st instar larvae 245-520. Since sex cannot be determined at larval stage, some of the mated female hostings were allowed to mature to adulthood then males and females were counted to determine the sex ratio. Early larvae showed an average of 82.9% females and late larvae had an average of 84.2%. Pupae collections were made among the progeny of mated females provided with hosts for 48 hrs. They were sorted by sex and stage (early yellow, red-eye, half black, and black pupae). Equal numbers (S20) of pupae from each stage were then pooled prior to RNA extraction. Males and females were extracted separately. Male reproductive tracts (testes, seminal vesicles, and accessory glands) were dissected in phosphate buffered saline (PBS) from and transferred with a small amount of PBS into a tube on dry ice. A total of 60 testes per replicate were dissected (10 each yellow/red, salt & pepper, and black pupal stages). Female reproductive tracts (30 per replicate) were removed from 1-3 days post eclosion virgin females and transferred to a tube on dry ice.
|
| Growth protocol |
51-61 hours post laying; 245-520 individuals per replicate; Male Samples are 100% male, Female Samples are on average 82.9 to 84.2 percent female based on sexing of individuals from matching
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was homogenized using Trizol reagent (Invitrogen) and extracted RNA was purified using the Qiagen RNeasy protocol with on-column DNase treatment from specific tissues. Total RNA amplified using MessageAmp II aRNA Kit (Ambion). aRNA converted to ds cDNA using SuperScript cDNA Synthesis Kit (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
DNA labeling using 1 O.D. CY-labeled random nonomer primer and 100U Klenow fragment (3>5 exo) per 1 μg double-stranded cDNA in triplicate.
|
| |
|
| Channel 2 |
| Source name |
1st instar female larva; 51-61 hours post laying; 245-520 individuals per replicate
|
| Organism |
Nasonia vitripennis |
| Characteristics |
Sex: female
developmental stage: 1st instar larva
tissue: whole body
strain: AsymCX
|
| Treatment protocol |
Samples were prepared at 25º C as follows: Approximately 200 N. vitrirpennis (AsymCX) virgins were collected as black pupa. When eclosed, half of them were provided with males and were allowed to mate overnight, and the other half were kept as virgins. Virgin setting result in all-male progeny whereas mated females produced ~89.5% female progeny under the design used here. Females were provided 15- 20 Sarchophaga bullata hosts in groups of 20 females for 24 hrs to induce production of eggs. The hosts were then removed and females were left overnight (~18 hrs). To collect embryos, individual females were given access to a host at one end (to restrict the oviposition site) and allowed to lay eggs for 6-10 hrs before being removed. Embryos were then harvested immediately (early embryos), 18 hrs later (late embryos), or 51 hrs later (1st instar larvae). All embryos and larvae were collected in an RNase-free environment. The host was cracked open and the "cap" removed to expose embryos. Dissecting needles were used to gently scrape embryos from the surface of the host and transfer them into a 1.5 ml tube pre-chilled on dry ice. Samples were stored at -80º C. If at anytime the host was punctured or embryos were exposed to host hemolymph, they were discarded. Estimates of the number of embryos per replicate (three per life stage/sex) were recorded; early embryos ranged from 300-900, late embryo 140-500, 1st instar larvae 245-520. Since sex cannot be determined at larval stage, some of the mated female hostings were allowed to mature to adulthood then males and females were counted to determine the sex ratio. Early larvae showed an average of 82.9% females and late larvae had an average of 84.2%. Pupae collections were made among the progeny of mated females provided with hosts for 48 hrs. They were sorted by sex and stage (early yellow, red-eye, half black, and black pupae). Equal numbers (S20) of pupae from each stage were then pooled prior to RNA extraction. Males and females were extracted separately. Male reproductive tracts (testes, seminal vesicles, and accessory glands) were dissected in phosphate buffered saline (PBS) from and transferred with a small amount of PBS into a tube on dry ice. A total of 60 testes per replicate were dissected (10 each yellow/red, salt & pepper, and black pupal stages). Female reproductive tracts (30 per replicate) were removed from 1-3 days post eclosion virgin females and transferred to a tube on dry ice.
|
| Growth protocol |
51-61 hours post laying; 245-520 individuals per replicate; Male Samples are 100% male, Female Samples are on average 82.9 to 84.2 percent female based on sexing of individuals from matching
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was homogenized using Trizol reagent (Invitrogen) and extracted RNA was purified using the Qiagen RNeasy protocol with on-column DNase treatment from specific tissues. Total RNA amplified using MessageAmp II aRNA Kit (Ambion). aRNA converted to ds cDNA using SuperScript cDNA Synthesis Kit (Invitrogen).
|
| Label |
Cy5
|
| Label protocol |
DNA labeling using 1 O.D. CY-labeled random nonomer primer and 100U Klenow fragment (3>5 exo) per 1 μg double-stranded cDNA in triplicate.
|
| |
|
| |
| Hybridization protocol |
Labeled products were pooled to serve as a Master Mix for Array set. The products were dried in a speedvac then resuspended in water prior to hybridization, which was performed with the NimbleGen hybridization kit. A single hybridization master for a given source and biological replicate
|
| Scan protocol |
Axon GenePix 4200 Professional, 5 micron resolution
|
| Description |
exp3_matrix_chip1.txt
|
| Data processing |
Raw signal intensity extract with NimbleScan 2.4 Software (Roche NimbleGen) in PAIR files. Quantile normalization to convert raw to processed data.
|
| |
|
| Submission date |
Apr 15, 2013 |
| Last update date |
Apr 15, 2018 |
| Contact name |
Justin Choi |
| E-mail(s) |
jeochoi@gmail.com
|
| Phone |
1-706-721-6757
|
| Organization name |
Georgia Regents University
|
| Department |
Cancer Center
|
| Street address |
1410 Laney Walker Blvd.
|
| City |
Augusta |
| State/province |
GA |
| ZIP/Postal code |
30912 |
| Country |
USA |
| |
|
| Platform ID |
GPL16800 |
| Series (2) |
| GSE44701 |
Nasonia Tiling Microarray Experiments for Expression Profiling Sexual Development |
| GSE46080 |
Nasonia Tiling Microarray Experiments for Expression Profiling Sexual Development (Larva) |
|
