Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

HALO-RPD: In Searching for RNA-Binding Protein Targets in Plants

Version 1 : Received: 22 July 2021 / Approved: 23 July 2021 / Online: 23 July 2021 (09:32:28 CEST)

How to cite: Shamustakimova, A. HALO-RPD: In Searching for RNA-Binding Protein Targets in Plants. Preprints 2021, 2021070531 (doi: 10.20944/preprints202107.0531.v1). Shamustakimova, A. HALO-RPD: In Searching for RNA-Binding Protein Targets in Plants. Preprints 2021, 2021070531 (doi: 10.20944/preprints202107.0531.v1).

Abstract

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding the RNA biology. Currently, various methods are available to investigate these interactions, in particular, RNA pulldown assay. In the present paper, a method based on the HaloTag technology is presented that is called Halo-RPD (HaloTag RNA PullDown). The proposed protocol uses plants with stable fusion protein expression and the MagneBeads magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic A. thaliana plants. The key stages described in the paper are as follows: 1) preparation of the magnetic beads 2) tissue homogenization and collection of control samples 3) precipitation and wash of RNA-protein complexes; 4) evaluation of protein binding efficacy; 5) RNA isolation; 6) analysis of the obtained RNA. Recommendations for better NGS assay designs are provided.

Keywords

A.thaliana; HaloTag; RNA-binding proteins; RNA pulldown assay; RNA-protein complexes; cold shock domain protein

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