E14 cells were infected with lentivirus expressing non-targeting shRNA (shCtrl) or shSin3a-1. After puromycin selection, E14 cells were collected and placed on ice in the Trizol solution (Invitrogen).
Growth protocol
Low passage (p9) E14 cells were maintained on plates coated with 0.1% gelatin in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 15% fetal bovine serum (Gibco), 1% penicillin and streptomycin (Gibco), 2 mM L-glutamine (Thermo), 100 mM nonessential amino acids (NEAA; Thermo), 100 μM β-mercaptoethanol (Gibco), and leukemia inhibitory factor (1000 U/ml LIF; Millipore).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions.
Label
biotin
Label protocol
Total RNA were amplified, labeled and purified by using GeneChip 3’IVT Express Kit (Cat#901229, Affymetrix, Santa Clara, CA, US) followed the manufacter’s instructions to obtain biotin labeled cRNA.
Hybridization protocol
Array hybridization and wash was performed using GeneChip Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instruction.
Scan protocol
Slides were scanned by GeneChip Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
Description
Gene expression data from ESCs infected with shCtrl virus as control
Data processing
Expression microarray of Affmetrix TVT analyses were performed using affy of Bioconductor, applying the MAS5 method for normalization. affymetrix-algorithm-name = ExpressionStat affymetrix-algorithm-version = 5.0 program-name = Expression Console program-id = 1.4.1.46
