cell type: J1 mouse embryonic stem cells treated with: RA (Retinoic acid 1μM) for 24hrs
Treatment protocol
The J1 mESCs were treated with a final concentration of RA (1μM), DMSO (1μL), VC (50μg), ddH2O (1μL)
Growth protocol
The J1 mESC was gelatin adapted and plated on 0.2% (w/v) gelatin coated tissue culture plates in Knockout DMEM supplemented with 15% (v/v) Knockout Serum Replacement, 1× non-essential amino acids, 100 μM β-mercaptoethanol, 2 mM glutamine, 50 units/ml Penicillin, 50 μg/ml Streptomycin, 1000 U/ml LIF at condition of 37℃ and 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total cellular RNA was isolated from J1 mESC using TRIzol RNA Isolation Reagent (Invitrogen, USA) following the manufacturer's recommendations. Total RNA was checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy mini kit (QIAGEN, GmBH, Germany) and RNase-Free DNase Set (QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit.
Description
RA_003
Data processing
The scanned images were analyzed with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
