Transcriptome survey of human gastric cancer cells with Tanshinone IIA treatment
Total RNA was extracted from AGS cells treated with DMSO (control) or TIIA, using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. The quantity and quality of RNA were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), and were found to have an RNA Integrity Number (RIN) value of more than 9. Poly(A) mRNA was isolated with oligo(dT)-bound magnetic beads and incubated with fragmentation buffer to form short RNA fragments. Reverse transcriptase and random hexamer primers were used to synthesize the first-strand cDNA, and DNA polymerase I and RNaseH were used to synthesize the second-strand cDNA. Double stranded cDNA was end-repaired with T4 DNA polymerase, Klenow fragments, and T4 Polynucleotide Kinase, after which a single “A” base was added, and the entire sequence was ligated to Illumina sequencing adapters (Illumina, San Diego, CA). PCR was performed to amplify the fragments. Using Illumina HiSeqTM 2000 (Illumina), the cDNA library was sequenced on a flow cell after validation on an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System.  After the base calling, the reads with adaptor sequences and low quality reads were removed. The low quality reads are defined as the reads with greater than 10% of unknown bases (N) or greater than 50% of low quality bases which quality values are less than 5. These high quality reads were mapped to hg19 human genome assembly using SOAPaligner in SOAP2 with 2 mismatch allowance and annotated based on the GENCODE. Read counts for individual GENCODE genes were subsequently determined using HTSeq-count (http://www-huber.embl.de/users/anders/HTSeq) by considering only uniquely mapped reads. The gene expression level of individual gene is calculated by using RPKM (Reads Per Kilobase per Million mapped reads) method. The differentially expressed genes were identified by Poisson distribution model.