cell line: HeLa condition: NT exposure time: 8 weeks rna integrity number: 10
Treatment protocol
One day after seeding, cells were exposed to 0.25 ng/ml of WT or HA CDT for 48 H. For chronic exposure, individual clones were selected, the rest of the cells were pooled. CDT was added at each passage (every 2-3 days) for 8 weeks.
Growth protocol
HeLa cells were were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotics (penicillin/streptomycin). Cells were plated at a density of 150000 cells per well in 6 wells plate
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) mRNA were extracted and the quality of these samples was assessed (Agilent RNA 6000 Nano Kit Quick, Agilent Bioanalyzer 2100); RNA Integrity Number (RIN) of these mRNA was superior to 9.8.
Label
Cy3
Label protocol
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE v2 microarray (8X60K, Design 039494) enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).
Scan protocol
Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software
Data processing
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies, Santa Clara, CA) using default parameters (protocol GE1_1010_Sep10 and Grid: 039494_D_F_20120628). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0; okFoundGreen<-x$gIsFound==1; okPos=x$gIsPosAndSignif==1; okWellAbove<- x$gIsWellAboveBG==1; as.numeric(okType & okFoundGreen & okPos & okWellAbove);} We selected the spots with a minimal weight of 1 for 12 out of 15 microarrays or with a minimal weight of 3 per group from at least one experimental group. At this step, 32772 spots out of 62976 were selected. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 26516 rows each corresponding to a unique ProbeName (provided as data Matrix).
