|
| Status |
Public on May 19, 2012 |
| Title |
LR_S20-13 LR_SAL_Lsal_S20ppt_Cop_Trial2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pool of whole copepodids
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
salinity: 20 ppt
temperature: 10ºC
|
| Treatment protocol |
Copepodids were pooled into groups of ~500 lice per beaker. Triplicate flasks were incubated for 24 hr at 4, 10, or 16ºC with salinity held constant at 30 ppt. In another experiment, triplicate flasks containing seawater diluted to 30, 25, 20, or 10 ppt were incubated at 10ºC. Both of these low resolution experiments were repeated once. A single trial of a high resolution salinity experiment was conducted as above, but with 6 beakers per condition and a salinity range of 30, 29, 28, 27, 26 and 25 ppt, and a constant temperature of 10ºC. The lice were recovered onto 47 mm cellulose acetate/cellulose nitrate filter membranes with a pore size of 8.0 μm. The membranes were flash frozen in liquid nitrogen and stored at -80ºC.
|
| Growth protocol |
L. salmonis obtained from seawater netpen-reared Atlantic salmon Salmo salar in coastal British Columbia, were maintained in cold aerated seawater during transport to the Pacific Biological Station, Nanaimo, BC. Intact and pigmented egg strings were removed and incubated in flasks containing 400ml of filtered and aerated seawater. The resulting nauplii were maintained at 30 ppt salinity until a majority moulted to copepodids.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen filters containing lice were homogenized, and extracted using TRIzol® (Invitrogen), as per manufacturers’ instructions. Total RNA was purified through RNeasy spin columns with an on-column DNase I treatment (QIAGEN) to degrade genomic DNA. Total RNA was then quantified by spectrophotometry (NanoDrop-1000), and quality checked by electrophoresis on a 1% agarose gel. Samples were then randomized for all downstream nucleic acid manipulations.
|
| Label |
Cy5-CTP
|
| Label protocol |
Purified total RNA (200ng) was reverse-transcribed to cDNA using Low Input Quick Amp Labeling kits (Agilent), as per manufacturer’s instructions for hybridization to a 4x44K oligo gene expression microarray. Labelled cRNA was then purified through RNeasy columns as per manufacturer’s instructions (QIAGEN) and quantified using spectrophotometry (NanoDrop-1000). For quality control, the specific activity of all samples was greater than 6 pmol dye per μg cRNA (Agilent). Samples were kept at -80ºC until hybridization.
|
| |
|
| Channel 2 |
| Source name |
Pool of samples from current experiment (from all LR_SAL conditions except 25 ppt)
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
sample type: reference
|
| Treatment protocol |
Copepodids were pooled into groups of ~500 lice per beaker. Triplicate flasks were incubated for 24 hr at 4, 10, or 16ºC with salinity held constant at 30 ppt. In another experiment, triplicate flasks containing seawater diluted to 30, 25, 20, or 10 ppt were incubated at 10ºC. Both of these low resolution experiments were repeated once. A single trial of a high resolution salinity experiment was conducted as above, but with 6 beakers per condition and a salinity range of 30, 29, 28, 27, 26 and 25 ppt, and a constant temperature of 10ºC. The lice were recovered onto 47 mm cellulose acetate/cellulose nitrate filter membranes with a pore size of 8.0 μm. The membranes were flash frozen in liquid nitrogen and stored at -80ºC.
|
| Growth protocol |
L. salmonis obtained from seawater netpen-reared Atlantic salmon Salmo salar in coastal British Columbia, were maintained in cold aerated seawater during transport to the Pacific Biological Station, Nanaimo, BC. Intact and pigmented egg strings were removed and incubated in flasks containing 400ml of filtered and aerated seawater. The resulting nauplii were maintained at 30 ppt salinity until a majority moulted to copepodids.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen filters containing lice were homogenized, and extracted using TRIzol® (Invitrogen), as per manufacturers’ instructions. Total RNA was purified through RNeasy spin columns with an on-column DNase I treatment (QIAGEN) to degrade genomic DNA. Total RNA was then quantified by spectrophotometry (NanoDrop-1000), and quality checked by electrophoresis on a 1% agarose gel. Samples were then randomized for all downstream nucleic acid manipulations.
|
| Label |
Cy3-CTP
|
| Label protocol |
Purified total RNA (200ng) was reverse-transcribed to cDNA using Low Input Quick Amp Labeling kits (Agilent), as per manufacturer’s instructions for hybridization to a 4x44K oligo gene expression microarray. Labelled cRNA was then purified through RNeasy columns as per manufacturer’s instructions (QIAGEN) and quantified using spectrophotometry (NanoDrop-1000). For quality control, the specific activity of all samples was greater than 6 pmol dye per μg cRNA (Agilent). Samples were kept at -80ºC until hybridization.
|
| |
|
| |
| Hybridization protocol |
Sample loading was performed as per manufacturers’ instructions using a 4x44k array and SureHyb chambers (Agilent). Arrays were hybridized at 65ºC for 17 hr at 10 rpm in a hybridization oven (Agilent). Washing was performed as per manufacturers’ instructions, using the optional protocol to prevent ozone degradation. All slides were transferred to a dark box and kept at low ozone until scanned.
|
| Scan protocol |
Arrays were scanned on a Perkin Elmer ScanArray Express at 5 μM resolution using PMT settings optimized to have the median signal of ~1-2% of array spots saturated (Cy5: 70; Cy3: 70).
|
| Description |
LR_SAL_Lsal_S20ppt_Cop_Trial2
|
| Data processing |
Images were quantified in Imagene 8.1 (Biodiscovery) using a custom eArray GAL file (Agilent). Poor spots and control spots were flagged by the software for downstream filtering. A block-specific background correction was performed by subtracting the average median signal for negative control spots from each signal median. Each experiment was normalized and filtered separately, using the following: raw values thresholded to 1; intensity-dependent Lowess normalization; and baseline transformation to the median of all samples. Control spots, and any entities not passing the following filter were removed from the analysis: raw values ≥ 500 in at least 65% of samples in any one condition; no flags in at least 65% of samples in any one condition.
|
| |
|
| Submission date |
May 18, 2012 |
| Last update date |
May 19, 2012 |
| Contact name |
Ben F Koop |
| E-mail(s) |
bkoop@uvic.ca
|
| Phone |
(250) 472-4067
|
| Organization name |
The University of Victoria
|
| Department |
Biology
|
| Lab |
Centre for Biomedical Research
|
| Street address |
PO Box 3020 STN CSC
|
| City |
Victoria |
| State/province |
BC |
| ZIP/Postal code |
V8W 3N5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL15566 |
| Series (1) |
| GSE37976 |
Transcriptomics of coping strategies in free-swimming Lepeophtheirus salmonis (Copepoda) |
|
