Work

Nuclear Transcriptomes at High Resolution Using Retooled INTACT

Year: 2017

Type: article

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 N uclei from TA gged specific C ell T ypes (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 E. 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 rRNA degradation procedure that minimizes 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-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. Summary: Improved technology and methodology for affinity purification of nuclei and analysis of nuclear transcriptomes, chromatin and other nuclear components.

Source: PLANT PHYSIOLOGY

DOI: 10.1104/pp.17.00688