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| Status |
Public on Nov 24, 2013 |
| Title |
A19C |
| Sample type |
SRA |
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| Source name |
Liver tissues, Cancer
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| Organism |
Homo sapiens |
| Characteristics |
paired sample id: A19
tissue type: Cancer
virus infection: HBV
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted from about 60 mg of tissues for each of the 10 paired samples using the mirVana™ miRNA Isolation Kit (Ambion Inc.) according to the manufacturer’s instruction. The RNA yields were quantified by NanoDrop ND1000 (Thermo- Fisher Scientific, Waltham, MA) and the RNA quality was assessed by the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). The RNA integrity number (RIN) of every RNA sample used for sequencing was more than 8. The cDNA libraries for 10 paired samples were constructed using mRNA-Seq Sample Prep Kit based on the Illumina Inc.’s guide. In brief, polyA-containing mRNA was purified using oligo-dT beads from 10ug of total RNAs for each sample and fragmented into small fragments using divalent cations under elevated temperature. The cleaved RNA fragments were reverse-transcribed into first strand cDNA using random primers (Invitrogen Inc.), followed by second-strand cDNA synthesis. After end-repair processing, a single ‘A’ base was added to cDNA fragments at 3' end. The cDNAs were then ligated to adapters, purified by 2% agrose gel, and then enriched by PCR to create the final cDNA library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
All alignments were performed using a tool package SOAP2, a program specially designed for next-generation sequencing analysis, allowing up to 2 mismatches. Sequenced reads were respectively aligned to human transcript reference sequences from ENSEMBL database (build 55) for expression evaluation at the level of gene or exon. Reads that unable to be mapped to transcriptome were also aligned to novel exon-exon junction database that was previously developed by our group for detection of new alternative splicing events.After alignment to the transcriptome, the expression level of genes was determined based on the value of RPKM (reads per kilobase per million), which was calculated as the number of reads mapped to the transcripts of one gene divided by the transcript length and the number of total mapped reads in one sample. The expression level of each exon or exon-exon junction was also evaluated by RPKM, similarly calculated as the number of reads mapped to unique exon divided by the exon length and the number of total mapped reads in one sample. Three kinds of statistical analyses were applied to estimate the significance of expression difference, including paired t-test and paired wilcoxon signed rank test based on RPKM value, and a recently proposed software package edgeR (a program specifically designed for analyzing RNA-seq data ) based on read count. The difference with P <0.05 for t test and wilcoxon signed rank test and FDR<0.05 for edgeR was considered as significant. To assess the distribution of DEGs at different expression abundance levels, MA-plot was made.
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| Submission date |
Nov 24, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Biaoyang Lin |
| Organization name |
ZheJiang University
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| Department |
ZCNI
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| Lab |
Systems Biology Division
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| Street address |
866 Yu Hang Tang Road Zi Jin Gang Campus Zhejiang University.
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| City |
HangZhou |
| ZIP/Postal code |
310058 |
| Country |
China |
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| Platform ID |
GPL9052 |
| Series (1) |
| GSE25599 |
RNA-seq analysis generated a comprehensive landscape of transcriptomes and revealed complex patterns of transcripts in Chinese HBV-related hepatocellular carcinoma |
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| Relations |
| SRA |
SRX032921 |
| BioSample |
SAMN00139545 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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