cell line: A549 treatment: transduced with pSuper Retro Puro shSMAD3 treated with vehicle for 12hr
Treatment protocol
Retro-viral vector was co-transfected with the pIK packaging plasmid in 293FT cells by using standard calcium phosphate transfection method. Thirty-six hours after the co-transfection, supernatants were collected and incubated with cells to be infected for 24 hours in the presence of polybrene (2.5 μg/mL). Following infection, puromycin (1.5μg/mL) was used to select stably transduced cells for 10 days.
Growth protocol
A549 cells were cultured in complete DMEM medium and supplied with 10% FBS.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using RNeasy RNA isolation kit (Qiagen) following supplier’s protocol.
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), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Label, hybridization and scanning was performed in shanghai biochip corporation (http://www.shbiochip.com/) following standard Agilent protocol.
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=5μm, PMT 100%, 10%, 16bit. Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US).
Description
A549 Cells transduced with pSuper Retro Puro shSMAD3 treated with vehicle for 12hr
Data processing
Data normalization and transformation was performed on Genespring GX 11 (Agilent) in shanghai biochip corporation.
