cell line: OE33 sirna transfection: ON-TARGETplus Non-targeting Pool
Treatment protocol
Cells were plated in wells in a 6 well-plate (0.2 million cells/well). 24 hours after plating, the cells were transfected using lipofectamin 2000 (Life Technologies, Grand Island, NY, USA) and siRNA (final concentration 50nM). The following siRNAs were used: MS_1 and MS_3 were transfected with ON-TARGETplus SMARTpool ARID1A (Thermo Fisher Scientific, Lafayette, CO, USA), whereas MS_2 and MS_4 were transfected with ON-TARGETplus Non-targeting Pool (Thermo Fisher Scientific). The cells were washed, harvested, and pelleted 48 hours after transfection.
Growth protocol
Cells were cultured in 1640 RPMI supplemented with 15% Fetal Bovine Serum
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the Rneasy kit (Qiagen, Germantown, MD, USA) according to the manufacturer's manual.
Label
Biotin
Label protocol
One μg total RNA was combined with 2 μl T7 oligo(dT) primer and 2 μl Poly-A controls and brought to a volume of 12 μl. The samples were incubated at 70ºC for 10 minutes to label as per standard Affymetrix protocol.
Hybridization protocol
The samples were ice quenched and combined with the hybridization cocktail (5 μl oligonucleotide B2 control, 15 μl 20X eukaryotic hybridization controls, 150 μl 2X hybridization buffer, 30 μl 100% DMSO and 70 μl water). After 10 minutes of pre-hybridizing Human PrimeView array at 45ºC, 60 rpm, 200 μl of cocktail was loaded onto each array and the arrays were hybridized for 16 hours at 45ºC, 60 rpm. The cocktail was removed and the arrays were stained and washed using the Affymetrix GeneChip Fluidics Station 450 and FS450_001 fluidics script.
Scan protocol
Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was perfomed through the Affymetrix GeneChip Command Console version 3.4 (AGCC v3.4) Affymetrix software.
Data processing
Data were extracted from the CEL files, RMA normalized and converted to Log2 notation with Partek Genomics Suite (Partek, Missouri, USA). These values were labeled as to ARID1A knockdown status and the groups compared with a standard 1-way ANOVA, Student's t-test.
