array scanning protocol
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
normalization data transformation protocol
Data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores will be included in the supplemental file. ID_REF = Unique identifiers from GPL6947-21367 Z_VALUE = Z transformation of the natural log of the raw intensity values Detection_Pval = Detection Pvalue from Illumina GenomeStudio software, ver 1.1.1. ID_REF = VALUE = Z transformation of the natural log of the raw intensity values Detection Pval =
Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix Human HT-12 ver3. Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has ~48,000 well-annotated RefSeq transcripts with approximately 15-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
sample treatment protocol
Both the highly invasive and poorly invasive cell lines were treated with y-irradiation, and RNA was taken at 1 hour, 24 hours and 5 days after irradiation. Control cells from each group were not treated with radiation and RNA was isolated after 24 hours.
nucleic acid extraction protocol
RNA was extracted from the cells of each time point using Trizol (Invitrogen) and an RNeasy Mini kit (Qiagen) according to the manufactures standard protocols.
FS cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The media was supplemented with 100 U/ml penicillin and streptomycin and 4 mM L-glutamine. All cells were cultured at 37°C in 5% CO2.