|
| Status |
Public on Jun 20, 2018 |
| Title |
LincU_KD_ESC_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 46C
genotype/variation: shLincU
|
| Treatment protocol |
mESCs were harvested after removing the feeder,and RNA was harvested using Trizol reagent
|
| Growth protocol |
The mESC line 46C was cultured in 15% serum + LIF (1,000 U/ml, Millipore) on a feeder
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNAiso (Takara) following the manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 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 |
0.6 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 22.5ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers' instructions. On completion of the fragmentation reaction, 22.5ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-074809 SurePrint G3 Mouse GE v2 8x60K Microarray 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 (G2505C) using one color scan setting for 4x180k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of lincU knockdown mESCs
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 13.0(Agilent). Probe that at least 1 out of 2 samples flagged as Detected were maintained.
|
| |
|
| Submission date |
Nov 28, 2017 |
| Last update date |
Jun 20, 2018 |
| Contact name |
Zeyidan Jiapaer |
| E-mail(s) |
zeyidan17@163.com
|
| Organization name |
Tongji University
|
| Street address |
No.1239, Siping Road
|
| City |
Shanghai |
| ZIP/Postal code |
200092 |
| Country |
China |
| |
|
| Platform ID |
GPL21163 |
| Series (2) |
| GSE107416 |
LincU Preserves Naive Pluripotency by Restricting ERK Activity in Embryonic Stem Cells [knockdown] |
| GSE107418 |
LincU Preserves Naive Pluripotency by Restricting ERK Activity in Embryonic Stem Cells |
|
