Human ht12 version 4 arrays

RNA was isolated using a miRNeasy Mini Kit (QIAGEN), and qRT-PCR was performed using TaqMan probes (Life Technologies) and SYBR Green primers. Genome-wide expression analysis was carried out on Illumina Human HT12 version 4 arrays. Microarray data sets GSE31056 and GSE2379 were downloaded from NCBI GEO. TCGA data were downloaded from https://tcga-data.nci.nih.gov/tcga/ and http://www.broadinstitute.org/tcga/home.

Gene expression analysis was carried out on Illumina Human HT12 version 4 arrays. All data analyses were carried out on R using Bioconductor packages67 (link). Raw intensity data from the array scanner were processed using the BASH68 (link) and HULK algorithms as implemented in the bead array package69 (link). Log2 transformation and quantile normalization of the data were performed across all sample groups. Differential expression analysis was carried out using the Limma package70 (link). Differentially expressed genes were selected using a P-value cutoff of <0.05 after application of FDR correction for multiple testing applied globally to correct for multiple contrasts. RNA was extracted from cells (HB2, SUM159) treated with control and GNG12-AS1 siRNAs (exons 1 and 7). The analysis was performed with six biological replicates for each cell line. cDNA synthesis, labelling and array procedure were conducted at the Genomic Facility at the CRUK CI. Pathway analysis was performed using Metacore.