GSE99122
Nuclear and cellular mRNA sequencing of 1 cm root tips of 7 days Oryza sativa seedlings
Plants dynamically regulate chromatin architecture, transcription and post-transcriptional processes in accordance with developmental programs and environmental cues. Isolated nuclei can 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 with an outer nuclear envelope-anchored domain in the Nuclear Tagging Fusion protein and codon optimization of E. coli BirA, combined in a single T-DNA construct. We also developed an 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. The comparison of nuclear and steady-state poly(A)+ transcript populations of seedling root tips confirmed the capture of pre-mRNA and exposed distinctions between the nuclear and total RNA pool. The improved INTACT plasmid configuration for monocots and accompanying methods provide access to chromatin and pre-mRNA, paving the way for monitoring nuclear transcriptome and epigenome dynamics of specific cell-types in rice and other crop species. Overall design: 8 samples, 1 condition (1X MS, 1 % [w/v] Sucrose, pH 5.8). 4 bioreplicates of 2 RNA pools (total mRNA, rRNA degraded nuclear RNA)