Identification of ARGONAUTE/Small RNA Cleavage Sites by Degradome Sequencing

Methods Mol Biol. 2017:1640:113-128. doi: 10.1007/978-1-4939-7165-7_7.

Abstract

The method described here enables the high-throughput identification of cleaved mRNA targets of ARGONAUTE/small RNA complexes. The protocol is based on a modified 5'-rapid amplification of cDNA ends combined with deep sequencing of 3' cleavage products of mRNAs. Poly(A) RNA is purified from the tissue of interest which is followed by a 5'-RNA adapter ligation. The ligated products are then reverse transcribed, amplified, and digested with MmeI. After gel separation, a 3' double-stranded DNA adapter is ligated to the fragments, which are then amplified and index labeled for the high-throughput sequencing of pooled degradome libraries. Sequencing datasets from pooled libraries can be analyzed with different bioinformatic approaches.

Keywords: ARGONAUTE; DNA library construction; MmeI digestion; Next-generation sequencing; PAGE purification; PARE; Poly(A) mRNA; mRNA degradome; miRNA target.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Argonaute Proteins / metabolism*
  • DNA, Complementary / genetics
  • Gene Library
  • Genomics / methods*
  • High-Throughput Nucleotide Sequencing / methods
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Plant Proteins / metabolism*
  • Plants / genetics
  • Plants / metabolism*
  • Polymerase Chain Reaction / methods
  • Protein Binding
  • RNA Cleavage*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA, Plant / genetics
  • RNA, Plant / metabolism*

Substances

  • Argonaute Proteins
  • DNA, Complementary
  • MicroRNAs
  • Plant Proteins
  • RNA, Messenger
  • RNA, Plant