Viral small RNA cloning and sequencing

Methods Mol Biol. 2011;721:107-22. doi: 10.1007/978-1-61779-037-9_6.

Abstract

At the current rate of technological progress, high-throughput sequencing of nucleic acids has become a commodity. These techniques are perfectly suitable for viral small RNAs sequencing and contribute to the understanding of many aspects of virus biology in the context of host-pathogen interaction. However, the generation of high quality data is still an issue and the preparation of small RNAs libraries that accurately reflect the viral siRNAs in the sample remains a challenge. In this chapter we describe how to clone and sequence libraries of viral small RNAs from infected insect samples (mosquito, drosophilidae, insect-derived cell lines).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cloning, Molecular / methods*
  • Computational Biology
  • DNA Primers / genetics
  • DNA Primers / metabolism
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Isotope Labeling
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics
  • Oligonucleotides / isolation & purification
  • Oligonucleotides / metabolism
  • Polymerase Chain Reaction
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / isolation & purification
  • RNA, Small Interfering / metabolism
  • RNA, Viral / chemistry
  • RNA, Viral / genetics*
  • RNA, Viral / isolation & purification
  • RNA, Viral / metabolism
  • Reverse Transcription
  • Sequence Analysis, RNA / methods*
  • Viruses / genetics*

Substances

  • DNA Primers
  • Oligonucleotides
  • RNA, Small Interfering
  • RNA, Viral
  • endodeoxyribonuclease PmeI
  • Deoxyribonucleases, Type II Site-Specific