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Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
- Source :
- Journal of Visualized Experiments : JoVE
- Publication Year :
- 2011
- Publisher :
- MyJove Corporation, 2011.
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Abstract
- Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina's genome analyzer performs sequencing of a large number (10(7)) of relatively short sequence reads (150 bp).The "paired end" approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly. One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described(1, 2) but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published(3, 4) but require microgram amounts of starting total RNA. Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing(5), where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined "T7LA" (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome(6) and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples(7).
- Subjects :
- Issue 56
Sequence analysis
General Chemical Engineering
De novo transcriptome assembly
Biology
Polymerase Chain Reaction
General Biochemistry, Genetics and Molecular Biology
DNA sequencing
high throughput sequencing
Adapter (genetics)
Gene expression
Genetics
Humans
RNA, Messenger
Molecular Biology
Illumina dye sequencing
mRNA-Seq
General Immunology and Microbiology
General Neuroscience
Gene Expression Profiling
RNA
Sequence Analysis, DNA
Gene expression profiling
Illumina-Seq
Subjects
Details
- Language :
- English
- ISSN :
- 1940087X
- Issue :
- 56
- Database :
- OpenAIRE
- Journal :
- Journal of Visualized Experiments : JoVE
- Accession number :
- edsair.doi.dedup.....c2563acb370342684d71ac7b94f65793