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An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.
Heyer, Erin E; Ozadam, Hakan; Ricci, Emiliano P; Cenik, Can; Moore, Melissa J.
Affiliation
  • Heyer EE; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Howard Hughes Medical Institute, University of Massachusetts Medical School, Wo
  • Ozadam H; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Howard Hughes Medical Institute, University of Massachusetts Medical School, Wo
  • Ricci EP; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Howard Hughes Medical Institute, University of Massachusetts Medical School, Wo
  • Cenik C; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Howard Hughes Medical Institute, University of Massachusetts Medical School, Wo
  • Moore MJ; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Howard Hughes Medical Institute, University of Massachusetts Medical School, Wo
Nucleic Acids Res ; 43(1): e2, 2015 Jan.
Article in En | MEDLINE | ID: mdl-25505164
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Gene Library / Sequence Analysis, RNA / High-Throughput Nucleotide Sequencing Language: En Journal: Nucleic Acids Res Year: 2015 Document type: Article Country of publication: United kingdom
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Gene Library / Sequence Analysis, RNA / High-Throughput Nucleotide Sequencing Language: En Journal: Nucleic Acids Res Year: 2015 Document type: Article Country of publication: United kingdom