cell line: HN17 cell stage: Tumor stage transfection type: Stable tissue: Cell culture treatment: treated every 24 hours with 4 uM 5-aza-2- deoxycytidine for up to 16 days
Treatment protocol
To inhibit DNMTs, WSU-HN17 and Hacat cells were treated every 24 hours with 4 μM 5-aza-2- deoxycytidine for up to 16 days for genomic demethylation (Sigma-Aldrich). Oligosynthesis siRNA specific to DICER1 (sc-40489) purchased from Santa Cruz biotechnology for transient tranfection, was dilute with RNASE free dH2O to reach 10μM. In a 12 well plate, seed 6 x 105 WSU-HN17 cells per well in 1 ml antibiotic-free normal growth medium supplemented with 10% FBS. In next day, perform transfection those siRNA at 100 nM with Lipofectamine 2000 reagent according to the manufacturer's instructions. Cell was collect at 48h after transfection step by Trizol reagent (Life technologies, Inc.) in order to collect RNA for further study. For WSU-HN31 si-LINE-1 stable cell , after 48 h of transfection with LINE-1 siRNA constructs, cell was switch to culture with 50 ug/ml Hygromycin B (Cat. No. 10 843 555 001, Roche Applied Science) in Dulbecco's modified Eagle's medium (Gibco BRL, Life Technologies, Pairly, UK) supplemented with 10% heat- inactivated fetal bovine serum (Sigma, St. Louis, MO, USA). Cell was collect by Trizol reagent (Life technologies, Inc.) to RNA extraction for further study.
RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with Deoxyribonulease I (DNase I), RNase-free (Fermentas) accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.
Label
biotin
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Description
replicate 1
Data processing
The data were normalised using cubic spline normalisation with Beadstudio program.
