Mrna seq preparation kit

For RNA-Seq library preparation, total RNA was first extracted using Trizol, as previously described,^{21 (link), 22 (link)} from control and ESCC tissues (n=3 each). mRNA was then isolated, fragmented and reverse-transcribed into the cDNA libraries with an mRNA-Seq preparation kit (Illumina, San Diego, CA, USA). Library quality was assessed on an Agilent Bioanalyzer 2100 (Agilent, Shanghai, China). The Illumina Hiseq2000 platform was used for single-end sequencing of cDNA libraries. Alignment of reads was performed using Tophat2 with the mm10 build of the mouse genome. Transcript assembly and differential expression genes were identified via Cufflinks software (Seattle, WA, USA).^{23 (link)} RNA-Seq data were analyzed using the CummeRbund package in R.^{24 (link)} Functional enrichment analysis of differential expression genes was performed using the DAVID Functional Annotation Clustering Tool. A heat map of differentially expressed genes involved in glutathione metabolism was generated using Heatmap Builder.^{25 (link)}

Total RNA was extracted from cells using the Trizol reagent (Invitrogen), and cDNA was synthesized from total RNA using the SuperScript III (Invitrogen) according to the manufacturer’s guidance. qPCR was performed using the Real-Time Quantitative PCR SYBR Green kit (Takara, Tokyo, Japan). Primer sequences used were β-actin forward 5′-AGGGGCCGGACTCGTCATACT-3′; β-actin reverse 5′-GGCGGCACCACCATGTACCCT-3′; YAP forward 5′-TAGCCC TGCGTAGCCAGTTA-3′; YAP reverse 5′-TCATGCTTAGTCCACTGTCTGT-3′; ALDH1A1 forward 5′-GCACGCCAGACTTACCTGTC-3′; ALDH1A1 reverse 5′-CCTCCTCAGTTGCAGGATTAAAG-3′. The relative expression of each gene was calculated using the 2^ΔΔCT method relative to the expression levels of β-actin. For RNAseq library preparation, mRNA was then isolated, fragmented, and reverse-transcribed into the cDNA libraries with an mRNAseq preparation kit (Illumina, San Diego, CA, USA). Library quality was determined using Agilent Bioanalyzer 2100 (Agilent). The Illumina Hiseq 2000 platform was used for single-end sequencing of cDNA libraries. Alignment of reads was performed using Tophat2 with the hg19 build of the human genome. Transcript assembly and differential expression genes were identified by using Cufflinks software (Seattle, WA, USA). A heat map of differentially expressed genes involved in glutathione metabolism was generated using R.