|
| Status |
Public on Nov 25, 2020 |
| Title |
BMECs, EEF1D-1357 shRNA, replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
BMECs, EEF1D-1357 shRNA
|
| Organism |
Bos taurus |
| Characteristics |
cell type: Bovine mammary epithelial cell (BMEC)
passage: fifth passage
treatment: EEF1D-1357 shRNA
genotype: EEF1D knockdown
|
| Treatment protocol |
Using the blocks of fresh mammary gland epithelial tissue collected from a lactating Holstein cow as the abovementioned, the primary BMECs were isolated, cultured and purified by tissue block culture and enzyme digestion method. The BMECs at the fifth passage were used for subsequent experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified, reversely transcribed into cDNA, and synthesized into cRNAs labeled with Cy3 using a Quick Amp Labeling Kit (Agilent).
|
| 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 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 |
Gene expression after RNAi with EEF1D-1357
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Nov 24, 2020 |
| Last update date |
Nov 25, 2020 |
| Contact name |
Shan Lin |
| E-mail(s) |
[email protected]
|
| Phone |
17610075203
|
| Organization name |
China Agricultural University
|
| Street address |
No.2 Yuan Mingyuan West Road
|
| City |
Beijing |
| ZIP/Postal code |
100193 |
| Country |
China |
| |
|
| Platform ID |
GPL11649 |
| Series (1) |
| GSE162088 |
EEF1D RNAi in Bovine mammary epithelial cell |
|
