|
| Status |
Public on Sep 26, 2022 |
| Title |
Hepatocyte_48h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Rat primary hepatocyte, 48h, replicate2
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell: primary hepatocyte
gender: male
weight: 320g
|
| Treatment protocol |
The treatment in our study in the different length of in vitro culture.
|
| Growth protocol |
Primary hepatocytes isolated from rat liver were resuspended in William's E medium supplemented with 10% FBS (GBICO), 1% ITS, dexamethasone, 1 X L-glutamine/ penicillin /streptomycin and plated onto collagen-I-coated plate at the density of 6× 105 cells/ml. After 3 hours, non-attached cells were washed away and the medium was replaced with William's E free of FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNasey Mini Kit (Qiagen p/n 74104) following the manufacturer's recommendations.
|
| Label |
cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-2305) according to the manufacturer's instructions, followed by RNeasy Mini Kit (Qiagen p/n 74104). Dye incorporation and cRNA yield were checked with theNanoDrop ND-2000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25ul of 2x GEx Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent Rat lncRNA Microarray V3 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 Agilent Microarray Scanner (Agilent p/n G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression at 48h in replicate2 of primary hepatocytes
|
| Data processing |
The scanned images were analyzed with Agilent Feature Extraction software 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.
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| |
|
| Submission date |
Sep 26, 2019 |
| Last update date |
Sep 26, 2022 |
| Contact name |
jiang zhengyi |
| E-mail(s) |
zhengyijiang@zju.edu.cn
|
| Phone |
15858111920
|
| Organization name |
Zhejiang University
|
| Street address |
Qingchun 79 Road
|
| City |
Hang Zhou |
| State/province |
State... |
| ZIP/Postal code |
310000 |
| Country |
China |
| |
|
| Platform ID |
GPL27537 |
| Series (1) |
| GSE138071 |
Integrative proteomics of primary hepatocytes during dedifferentiation |
|
