cell line: E14T cell type: mouse embryonic stem cells genotype/variation: overexpression of Ssbp3 for 96 hrs
Treatment protocol
Duplicated biological mouse ES cell samples transfected with Vector or Ssbp3 plasmid respectively for 96 hours were collected for gene expression profiling.
Growth protocol
Mouse E14T ES cells were cultured under feeder-free condition in GMEM (Glasgow's Minimum Essential Medium, Invitrogen) supplemented with 10% FBS (Fetal Bovine Serum, Hyclone), 1 mM sodium pyruvate (Sigma), 0.1 mM NEAA (Non-Essential Amino Acids, Invitrogen), 2 mM L-glutamine (Invitrogen), 100 μM β-mercaptoethanol (Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin (Hyclone), 1000 U/mL recombinant LIF (Leukemia Inhibitory Factor, Chemicon).
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 integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
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 manufacturer’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 instructions.
Scan protocol
Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with default settings.
Description
SSbp3-2 24/7
Data processing
Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
