Format

Send to

Choose Destination
Plant Physiol. 2018 Jan;176(1):270-281. doi: 10.1104/pp.17.00688. Epub 2017 Sep 27.

Nuclear Transcriptomes at High Resolution Using Retooled INTACT.

Author information

1
Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, California 92521.
2
Department of Plant Biology, University of California, Davis, California 95616.
3
Genome Center, University of California, Davis, California 95616.
4
IPS2, Institute of Plant Science-Paris Saclay (CNRS-INRA), University of Paris-Saclay, F-911405, Orsay, France.
5
Department of Biology, Emory University, Atlanta, Georgia 30322.
6
Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, California 92521 julia.bailey@ucr.edu.

Abstract

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here, we describe transfer of the isolation of nuclei from tagged specific cell types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear-envelope-targeting domain of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a ribosomal RNA degradation procedure that minimizes pre-ribosomal RNA (pre-rRNA) transcripts. A high-resolution comparison of nuclear and steady-state poly(A)+ transcript populations of seedling root tips confirmed the capture of pre-messenger RNA (pre-mRNA) and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell types of rice and other species.

PMID:
28956755
PMCID:
PMC5761756
DOI:
10.1104/pp.17.00688
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center