|
| Status |
Public on Dec 31, 2014 |
| Title |
Fertilized_Eggs_Female12_dyeswap_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Fertilized egg_high quality female
|
| Organism |
Gadus morhua |
| Characteristics |
tissue: fertilized egg
developmental stage: 7h post-fertilization (7 hpf, ~ 2-cell stage)
egg harvested from: high quality female
|
| Growth protocol |
Eggs and sperm were harvested by stripping and collected in plastic collection beakers. Eggs were transferred into 1.5 L graduated glass ‘fertilization’ beakers by gentle pouring and sperm was added using a plastic transfer pipette. The egg and sperm mixture was gently stirred using the pipette and 100 ml UV-treated, filtered seawater was added and again stirred with the pipette. The mixture was left to incubate for 1 minute after which 500 ml UV-treated, filtered seawater was added and left to incubate for 5 minutes. After this incubation, the fertilization beaker was filled to 1.4 L mark with UV-treated, filtered seawater, placed on the bench in the walk-in cold room (6°C) and left undisturbed until the embryos reached first cleavage (2-cell stage), approximately 7 hours post-fertilization (hpf). At first cleavage stage, 7hpf embryo samples were collected from the 'fertilization beaker'. A sterile transfer pipette was used to fill three 1.5 ml RNase-free tubes with 0.25 ml of floating eggs from the ‘fertilization beaker’. Seawater was removed by pipette, and samples were flash frozen in liquid nitrogen and stored at -80 °C until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Per female, 3 batches of 0.25 ml of eggs were homogenized in TRIzol (Invitrogen) Bio-Gen PRO200 tissue homogenizer (PRO Scientific Inc., Oxford, CT) equipped with a 5 mm x 150 mm generator tip, at speed setting 2-3 for 30 seconds or until no solids were visible. The homogenate samples were passed through QIAshredder columns (Qiagen) following the manufacturer’s instructions. Total RNA was extracted from the TRIzol homogenate according to the manufacturers’ instructions, and the triplicate extractions were pooled for each individual female. Total RNA was treated with DNAse I (Qiagen) to remove genomic DNA, and then purified using the RNeasy MinElute Cleanup kit (Qiagen) according to the manufacturer’s instructions.
|
| Label |
AlexaFluor 647
|
| Label protocol |
5 μg of DNAse-treated and column-purified total RNA was labeled using the SuperScript Direct cDNA Labeling kit (Invitrogen) according to the manufacturer's protocol. Each RNA was labeled in 4 labeling reactions: two with AlexaFluor 647 and two with AlexaFluor 555.
|
| |
|
| Channel 2 |
| Source name |
Fertilized egg_low quality female
|
| Organism |
Gadus morhua |
| Characteristics |
tissue: fertilized egg
developmental stage: 7h post-fertilization (7 hpf, ~ 2-cell stage)
egg harvested from: low quality female
|
| Growth protocol |
Eggs and sperm were harvested by stripping and collected in plastic collection beakers. Eggs were transferred into 1.5 L graduated glass ‘fertilization’ beakers by gentle pouring and sperm was added using a plastic transfer pipette. The egg and sperm mixture was gently stirred using the pipette and 100 ml UV-treated, filtered seawater was added and again stirred with the pipette. The mixture was left to incubate for 1 minute after which 500 ml UV-treated, filtered seawater was added and left to incubate for 5 minutes. After this incubation, the fertilization beaker was filled to 1.4 L mark with UV-treated, filtered seawater, placed on the bench in the walk-in cold room (6°C) and left undisturbed until the embryos reached first cleavage (2-cell stage), approximately 7 hours post-fertilization (hpf). At first cleavage stage, 7hpf embryo samples were collected from the 'fertilization beaker'. A sterile transfer pipette was used to fill three 1.5 ml RNase-free tubes with 0.25 ml of floating eggs from the ‘fertilization beaker’. Seawater was removed by pipette, and samples were flash frozen in liquid nitrogen and stored at -80 °C until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Per female, 3 batches of 0.25 ml of eggs were homogenized in TRIzol (Invitrogen) Bio-Gen PRO200 tissue homogenizer (PRO Scientific Inc., Oxford, CT) equipped with a 5 mm x 150 mm generator tip, at speed setting 2-3 for 30 seconds or until no solids were visible. The homogenate samples were passed through QIAshredder columns (Qiagen) following the manufacturer’s instructions. Total RNA was extracted from the TRIzol homogenate according to the manufacturers’ instructions, and the triplicate extractions were pooled for each individual female. Total RNA was treated with DNAse I (Qiagen) to remove genomic DNA, and then purified using the RNeasy MinElute Cleanup kit (Qiagen) according to the manufacturer’s instructions.
|
| Label |
AlexaFluor555
|
| Label protocol |
5 μg of DNAse-treated and column-purified total RNA was labeled using the SuperScript Direct cDNA Labeling kit (Invitrogen) according to the manufacturer's protocol. Each RNA was labeled in 4 labeling reactions: two with AlexaFluor 647 and two with AlexaFluor 555.
|
| |
|
| |
| Hybridization protocol |
Arrays were prehybridized at 42°C in Nexterion Oligo Prehyb Buffer (Schott Nexterion) for at least 2 hours. Arrays were washed at room temperature for with 0.1% SDS for 5 minutes, followed by 3 5-minute washes with water. Slides were dried by spinning them for 5 minutes at 200x g and kept at 42°C. LifterSlips were prepared by washing them sequentially with water, water, 70% ethanol and 100% ethanol and drying them using compressed air. For each array two labeled cDNAs were combined (one sample, one reference) and 2 μl of LNA blocker (Genisphere) and 40 μl of prewarmed 2x formamide-based hybridization buffer (Genisphere) was added. These samples were incubated at 80°C for 10 minutes and then kept at 42°C. Pre-warmed arrays were put in Corning hybridization chambers, LifterSlips were applied and the samples were loaded under the LifterSlips. Hybridizations were performed overnight for ~16 hours in a water bath at 42°C. After hybridization, arrays were washed at room temperature for 15 minutes with 2xSSC/0.2% SDS which was pre-warmed at 42°C. Next slides were washed at room temperature for 15 minutes with 2xSSC, followed by 2 15-minute washes with 0.2xSSC. Slides were dried by spinning them for 5 minutes at 200x g.
|
| Scan protocol |
Arrays were scanned at a resolution of 5 μm using a ScanArray Gx Plus scanner and ScanExpress v4.0 software (Perkin Elmer).
|
| Description |
dye-swap
female12_ds_2
|
| Data processing |
Signal intensity data was extracted from tiff images using Imagene v7.0 (BioDiscovery). In addition to automated quality flagging, spots affected by dust particles, scratches and other local artefacts were flagged manually. Raw data was read into Bioconductor package 'marray', base 2 logratios were calculated from background-corrected median signals and control spots were removed. Data was normalized using printtip loess. Logratio data for a particular spot was removed when the raw signal value in one of the channels of that spot was lower than the threshold, which was calculated as (average background + 2 * SD) for each channel on each array. Logratio data for a particular spot was also removed if the spot was flagged in Imagene. Logratios of duplicate spots were averaged, and logratios for the dye-swap arrays were multiplied by -1 to give all logratios the same orientation.
|
| |
|
| Submission date |
Jan 21, 2014 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Matthew Rise |
| E-mail(s) |
mrise@mun.ca
|
| Phone |
7098647478
|
| Organization name |
Memorial University of Newfoundland
|
| Department |
Department of Ocean Sciences
|
| Street address |
1 Marine Lab Road
|
| City |
St. John's |
| State/province |
NL |
| ZIP/Postal code |
A1C5S7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10532 |
| Series (1) |
| GSE54233 |
Variation in embryonic mortality and maternal transcript expression among Atlantic cod (Gadus morhua) females: a functional genomics study |
|
