Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and SMAD4 Y95 Mutation Transcriptomes. Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and SMAD4 Y95 Mutation Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of Y95 phosphorylation on SMAD4-mediated transcription in response to TGF-b signals. The goals of this study are to compare transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: mRNA profiles of HaCaT parental and SMAD4 KO cells stably expressing wild-type SMAD4 (WT), Y95E or Y95F mutant, and GFP (as control) under with or without TGF-b were generated by deep sequencing, using Illumina HiSeq X Ten. qRT–PCR validation was performed using TaqMan and SYBR Green assays Conclusions: hierarchical clustering of the global gene expression profiles of TGF-β treated cells revealed that wild-type-SMAD4- and Y95F-mutant-rescued SMAD4-null cells were highly similar to the parental HaCaT cells, whereas GFP- and Y95E-mutant-rescued cells formed a separate cluster Overall design: Wild Type and SMAD4 Y95 Mutation