strain: 129S4/SvJae tissue: Inner Cell Mass cell type: Embryonic Stem Cell treatment: 24h in 1mM AICAR
Treatment protocol
J1 mESCs maintained in medium containing 1000 U/mL LIF and treated by 3 μΜ CHIR99021 or 1mM AICAR for 24h.
Growth protocol
The J1 mouse embryonic stem cell line purchased from ATCC (Manassas, VA, USA) was gelatin adapted and grown 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 (ESGRO, Millipore, USA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US)following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=3μm, 20bit.
Description
Gene expression after 24h in AICAR treated J1 mESCs
Data processing
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).
