cell line: HBL100 cell type: human breast cancer cells genotype/variation: HIC1 knockdown
Treatment protocol
For restoring expression of HIC1 in MDA-MB-231 cells, human full-length HIC1 cDNA was inserted into lentivirus vector pHR-SIN-CSIGW. For the production of lentivirus, lenti-x cells were transfected with the PMD2.G, PSPAX2 and HIC1 expression vector using the lipofectamine 2000 (Invitrogen). After 48 hours, culture supernatants were collected and passed through 0.45µm filters, mixed with fresh media (1:1) and polybrene (8μg/ml) to infect target cells. For generation of a stable HIC1 knockdown cell line, GV248 lentiviral vectors expressing short hairpin RNAs targeting HIC1 were purchased from GeneChem Company (China, Shanghai). Lentiviruses were produced as described above and infected cells were selected by puromycin treatment at 1μg/ml.
Growth protocol
Human breast cancer cell lines MDA-MB-231 and the immortalized epithelial cells HBL-100 were maintained in DMEM (Hyclone), supplement with 10% FBS (GIBCO) and 1% penicillin/streptomycin. Cells were cultured at 37℃ water-saturated 5% CO2 atmosphere.
Extracted molecule
total RNA
Extraction protocol
Total RNA was checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
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), followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions.
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. Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) Raw data were normalized by Quantile algorithm, Gene Spring Software 12.0 (Agilent technologies, Santa Clara, CA, US).
Description
CGCD_KD Gene expression in HBL100 cell line, H1C1 konck-downed
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software 12.0 (Agilent technologies, Santa Clara, CA, US).
