Computational protocol: Integrating transcriptomics and proteomics to show that tanshinone IIA suppresses cell growth by blocking glucose metabolism in gastric cancer cells

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Protocol publication

[…] 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 scores were removed. The reads with low quality scores 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. The reads with high quality scores were mapped to the human reference genome hg19 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. Expression of each individual gene was quantified by using the Reads Per Kilobase per Million mapped reads (RPKM) method. The differentially expressed genes were identified by Poisson distribution model []. The sequences reported in this study have been deposited in the Sequence Read Archive database with accession number SRP049450. [...] We used MetaCore version 6.18 (GeneGo, St. Joseph, MI, USA) to perform a gene function analysis and to enrich the functional networks of identified genes collected from RNA-seq. There were 16,110 of 16,603 identified genes eligible for network enrichment via GO Processes and Metabolic Networks analysis. For analysis of differentially expressed genes (|log2(fold–change)| > 1), 2,717 of 2,761 DEGs were eligible for GO Processes enrichment analysis, and displayed classified genes (Table ). All enrichment analysis was tested using the p-value threshold p < 0.0001 for the data inputs. […]

Pipeline specifications

Software tools SOAPaligner, HTSeq, MetaCore
Databases SRA GENCODE
Organisms Salvia miltiorrhiza
Diseases Deficiency Diseases, Neoplasms, Stomach Neoplasms, Glucose Metabolism Disorders
Chemicals Adenosine Triphosphate, Glucose, Glucose-6-Phosphate, Lactic Acid