An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

Erin E Heyer; Hakan Ozadam; Emiliano P Ricci; Can Cenik; Melissa J Moore

doi:10.1093/nar/gku1235

An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

Nucleic Acids Res. 2015 Jan;43(1):e2. doi: 10.1093/nar/gku1235. Epub 2014 Dec 12.

Authors

Erin E Heyer¹, Hakan Ozadam¹, Emiliano P Ricci¹, Can Cenik², Melissa J Moore³

Affiliations

¹ 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, Worcester, MA 01605, USA.
² 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, Worcester, MA 01605, USA Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ 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, Worcester, MA 01605, USA [email protected].

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't
Validation Study

MeSH terms

DNA, Circular / chemistry
DNA, Single-Stranded / chemistry
Electrophoresis, Polyacrylamide Gel
Gene Library*
High-Throughput Nucleotide Sequencing / methods*
MicroRNAs / chemistry
Reverse Transcriptase Polymerase Chain Reaction
Sequence Analysis, RNA / methods*

Substances

DNA, Circular
DNA, Single-Stranded
MicroRNAs

Associated data

GEO/GSE63606

Grants and funding

Howard Hughes Medical Institute/United States