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Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
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Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

DOI:
10.3791/3340-v

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14:49 min

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October 27, 2011

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, , , , , ,
1Regenerative Biology,Morgridge Institute for Research, 2Department of Cell & Regenerative Biology,University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology,University of California

Chapters

  • 00:05Title
  • 02:02Procedural Flow Chart
  • 03:19cDNA Synthesis
  • 06:31End Repair, Paired End Library
  • 07:37Adding ‘A’ Bases to the 3′ End of the DNA Fragments
  • 08:31Adapter Ligation
  • 10:32Size Selection/Gel Purification
  • 11:20Evaluation of Single-Read and Paired-End Sequencing Libraries
  • 14:18Conclusion

Summary

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Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Tags

MRNA-SeqIllumina LibrariesGlobal Gene ExpressionMutation AnalysisAllele-specific ExpressionGenome-wide AnalysesNon-sequenced GenomesTranscript Copy NumbersIllumina’s Genome AnalyzerPaired End SequencingAlternate Splice JunctionsInsertions And DeletionsDe Novo Transcriptome AssemblyLimited Starting MaterialNanograms Of Total RNAPCR AmplificationMinimal PCR Amplification

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