High frequency of B-catenin mutations in mouse HCCs induced by a non-genotoxic CAR agonist

Microarray data analysis For the gene expression profile, 150 ng of RNA were amplified (Illumina TotalPrep RNA Amplification Kit), labeled and hybridized on Illumina microarray (RatRef-12 V1 BeadChips, Illumina Inc., San Diego, CA, USA), including 21,791 genes.The intensity files were loaded into the Illumina BeadStudio 3.0.19.0 software (Illumina Inc, San Diego, CA, USA) and BRB Array Tools (Version 4.2.0) for quality control and gene expression analysis. First, the quantile normalization algorithm was applied on the dataset. Only genes whose expression differed by at least 1.5 fold from the median in at least 20% of the arrays and characterized by a 50th percentile of intensities greater than 300 were retained. The FDR-adjusted p-values were calculated using the Benjamini-Hochberg procedure [39]. According to these criteria, 2,281 expressed transcripts out of 21,791 showed reproducible up- or down-regulation. To identify the differentially expressed genes, we applied the Random-Variance Model and Multivariate Permutation Test. Following this analysis, 1,784 genes showed reproducible up- or down-regulation in at least one comparison微阵列数据分析。针对基因表达谱分析，取150 ng RNA采用Illumina TotalPrep RNA扩增试剂盒进行扩增、标记，随后在Illumina微阵列（microarray，RatRef-12 V1 微珠芯片（BeadChips），美国加利福尼亚州圣地亚哥Illumina公司）上完成杂交，该微阵列涵盖21791个基因。将信号强度文件导入Illumina BeadStudio 3.0.19.0软件（美国加利福尼亚州圣地亚哥Illumina公司）与BRB阵列工具（BRB Array Tools，版本4.2.0）以开展质量控制与基因表达分析。首先对数据集应用分位数归一化（quantile normalization）算法，仅保留在至少20%的阵列中表达量与中位数差异至少达1.5倍，且信号强度的50百分位数大于300的基因。采用Benjamini-Hochberg法[39]计算错误发现率（False Discovery Rate，FDR）校正后的P值。按照上述筛选标准，21791个基因中共计2281个表达转录本呈现出可重复的上调或下调表达趋势。为鉴定差异表达基因，本研究采用随机方差模型（Random-Variance Model）与多元置换检验（Multivariate Permutation Test）开展分析。经上述分析后，共计1784个基因在至少一组对比中呈现出可重复的上调或下调表达。