Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq

Mauricio A Reynoso; Germain C Pauluzzi; Sean Cabanlit; Joel Velasco; Jérémie Bazin; Roger Deal; Siobhan Brady; Neelima Sinha; Julia Bailey-Serres; Kaisa Kajala

doi:10.21769/BioProtoc.2458

Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq

Bio Protoc. 2018 Apr 5;8(7):e2458. doi: 10.21769/BioProtoc.2458.

Authors

Affiliations

¹ Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, CA, USA.
² IPS2, Institute of Plant Science-Paris Saclay (CNRS-INRA), University of Paris-Saclay, Orsay, France.
³ Emory University, Atlanta, GA, USA.
⁴ Department of Plant Biology, UC Davis, Davis, CA, USA.
⁵ Genome Center, UC Davis, Davis, CA, USA.

Abstract

Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would not capture non-polyadenylated pre-mRNA, we also present our method to remove ribosomal RNA from the total nuclear RNA in preparation for nuclear RNA-Seq.

Keywords: Gene expression; INTACT; Nuclear purification; RNA extraction; RNA-Seq; rRNA degradation.