title: An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.
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.
authors.affiliation: 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.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Heyer
authors.affiliation: 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.
authors.forename: Hakan
authors.initials: H
authors.lastname: Ozadam
authors.affiliation: 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.
authors.forename: Emiliano P
authors.initials: EP
authors.lastname: Ricci
authors.affiliation: 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.
authors.forename: Can
authors.initials: C
authors.lastname: Cenik
authors.affiliation: 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 melissa.moore@umassmed.edu.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Moore
mesh_terms: D004270:DNA, Circular / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D035683:MicroRNAs / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D004270:DNA, Circular; D004277:DNA, Single-Stranded; D035683:MicroRNAs