Chemically synthesized Accell siRNAs (Dharmacon) were passively transfected into L-1236 cells using Accell delivery media (Dharmacon) and 1% heat inactivated FBS, according to the manufacturer’s instructions. Normal culture conditions were reestablished one day prior to harvesting the cells. Days of siRNA treatment are referred to as ‘x+y’, where ‘x’ equals the number of days when cells were treated with Accell siRNA and ‘y’ means the number of days after reestablishing standard FBS conditions. For each target, the shortest period of siRNA incubation was combined with the lowest siRNA concentrations necessary to obtain efficient knockdowns. The following conditions were used: NFKB1 (3+1 days, 1 µM), NFKB2 (2+1 days, 500 nM), RELA (2+1 days, 500 nM), RELB (3+1 days, 1 µM).
Growth protocol
Cells were cultivated in 90% RPMI 1640 (Gibco) + 10% heat inactivated FBS.
Extracted molecule
total RNA
Extraction protocol
Following RNAi treatment, total RNA from L-1236 cells was prepared accordingly to manufacturer’s protocol (RNeasy Kit; QIAGEN, Hilden, Germany). RNA quality was assessed by 260/280 and 260/230 ratios, as well as by RNA Integrity Number (RIN) using a Eukaryote total RNA nano chip in an Agilent 2100 Bioanalyser (Agilent Technologies). Only RNA samples with 260/280 and 260/230 ratios higher than 1.8, and a RIN higher than 8.5, were used for preparation of microarray samples.
Label
biotin
Label protocol
Microarray experiments were carried out following Ambion (Ambion, AMB), and Affymetrix (Affymetrix Inc, Santa Clara, CA) protocols. Briefly, 100 ng total RNA from each sample was reverse transcribed to cDNA, which was further amplified by in vitro transcription. Single stranded cDNA was then fragmented and labeled with biotin.
Hybridization protocol
Biotinylated cDNAs were hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays according to the Affymetrix protocol (Affymetrix Inc, Santa Clara, CA).
Scan protocol
Scanning was performed according to the Affymetrix protocol (Affymetrix Inc, Santa Clara, CA).
Description
Group1_Canonical_6
Data processing
For each experimental group (canonical or non-canonical) the data was normalized separately using RMA with background correction and quantile normalization as implemented in the Bioconductor library oligo. Finally the data was log2 transformed.
