Transfection of non-target control vector or vector expression shFoxm1, shMELK, and EZH2
Growth protocol
All cells were cultured using DMEM/F12(Lonza) supplemented with B27(Invitrogen), epidermal growth factor (EGF, 20 ng/ml; R&D Systems), basic fibroblast growth factor (bFGF, 20 ng/ml; R&D Systems), Heparin(5mg/ml; Sigma) in suspension.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the Qiagen RNeasy kit (Qiagen), including DNase I treatment before the final elution to eliminate any DNA contamination, following the manufacturer’s directions.
Label
biotin
Label protocol
A 100 ng aliquot of total RNA from four independent incubations was amplified using the GeneChip® WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA) following the manufacturer’s instructions and then 5.5 ug of cDNA was fragmented and terminally labeled.
Hybridization protocol
Labeled targets were hybridized to Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) for 16 h at 45°C and washed according to Affymetrix standard protocols.
Scan protocol
The Genechips were scanned with an Affymetrix G7 scanner.
Data processing
For gene expression analysis, arrays were normalized using RMA as implemented in Partek Genomics Suite 6.6 (Partek Incorporated, St Louis MO).
