Next Generation Sequencing (RNA-Sequencing) for the analysis of RUNX3 and YAP1 targets in TGFb-stimulated HEK 293 cells
Comparision of the gene expression profiles of the TGFb-stimulated and si-Con, si-RUNX3 and si-YAP1 treated cells. Overall design: Methods: HEK293 cells were treated with control siRNA (si-con), RUNX3-specific siRNA (si-RUNX3), or YAP1-specific siRNA (si-YAP1), and then stimulated with TGFß1. RNA was extracted from the cells at the indicated time points, and gene expression patterns were analyzed by mRNA sequencing (n=3; biological replicates). Total RNA was isolated using Trizol reagent (Invitrogen). RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands) and RNA quantification was performed using a ND-2000 Spectrophotometer (Thermo Inc., DE, USA). Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK). Isolation of mRNA was performed using the Poly(A) RNA Selection Kit (LEXOGEN, Inc., Austria). The isolated mRNAs were used for cDNA synthesis and shearing in accordance with the manufacturer's instructions. Indexing was performed using the Illumina indexes 1–12. The enrichment step was carried out using PCR. Subsequently, libraries were checked using the TapeStation HS D1000 Screen Tape (Agilent Technologies, Amstelveen, The Netherlands) to evaluate the mean fragment size. Quantification was performed using the library quantification kit and a StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA). Quality control of raw sequencing data was performed using FastQC. Adapters and low-quality reads (<Q20) were removed using FASTX_Trimmer and BBMap. Then, the trimmed reads were mapped to the reference genome using TopHat. The Read Count data were processed based on a FPKM + Geometric normalization method using EdgeR within R. FPKM (Fragments Per kb per Million reads) values were estimated using Cufflinks. Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc., Seoul, Korea).