Identification of ARGONAUTE/small RNA cleavage sites by degradome sequencing

Ivett Baksa, G. Szittya

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages113-128
Number of pages16
DOIs
Publication statusPublished - Jan 1 2017

Publication series

NameMethods in Molecular Biology
Volume1640
ISSN (Print)1064-3745

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Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Baksa, I., & Szittya, G. (2017). Identification of ARGONAUTE/small RNA cleavage sites by degradome sequencing. In Methods in Molecular Biology (pp. 113-128). (Methods in Molecular Biology; Vol. 1640). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7165-7_7