|
| Status |
Public on Apr 12, 2019 |
| Title |
PeriparturientNeu,C+7, rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Neutrophils_C+7
|
| Organism |
Bos taurus |
| Characteristics |
breed: Holstein Friesian
Sex: female
status: periparturient cows
time point: 7 d relative to actual calving date
tissue: Blood sampled from the jugular vein
cell type: Neutrophils
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was done using trizol according to manufacturer’s instruction (Ambion®). The appropriate precautions were used to avoid RNase contamination throughout in the entire procedure. Half (0.5) mL of 100% of isopropanol was added to each tube 114 containing the homogenized cell pellets in the TRIzol® Reagent and incubated at room temperature for 10 minutes. It was then centrifuged at 12,000× g for 10 minutes at 4 °C. Supernatant was removed from the tube leaving only the RNA pellet. The pellet was washed with 1 mL of 75% ethanol. The sample was vortexed briefly, and centrifuged at 7500×g for 5 minutes at 4°C. Supernatant was discarded and washed. RNA pellet was air dried for 10 minutes. The RNA pellet was resuspended in 30 µl RNase free water by passing the solution up and down several times through a pipette tip. RNA was stored at –80 °C. RNA concentration was measured using a Nano-Drop ND-1000 spectrophotometer (Nano-Drop Technologies). The purity of RNA (A260/A280) for all samples was above 1.80. The quality of RNA was evaluated using the Agilent Bioanalyzer system (Agilent 2100 Bioanalyzer, Agilent Technologies). The average RNA integrity number (RIN) value for samples was 9.0 ± 0.5
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
NeuC+7_002
Gene expression seven days post-calving cow neutrophils
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Apr 11, 2019 |
| Last update date |
Apr 12, 2019 |
| Contact name |
Mulumebet Worku |
| Organization name |
North Carolina Agricultural and Technical State University
|
| Department |
Animal Sciences
|
| Lab |
Genomic diversity and animal biotechnology
|
| Street address |
1601 E market Street
|
| City |
Greensboro |
| State/province |
North Carolina |
| ZIP/Postal code |
27411 |
| Country |
USA |
| |
|
| Platform ID |
GPL11648 |
| Series (1) |
| GSE129639 |
Global gene expression analysis of neutrophils from Periparturient Holstein Cows |
|
