Computational protocol: Surveillance of 3′ Noncoding Transcripts Requires FIERY1 and XRN3 in Arabidopsis

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Protocol publication

[…] Wild-type, fry1-6, xrn3-3, xrn2-1xrn4-6, and xrn3-3xrn4-6 were grown in Linsmaier and Skoog (LS; Caisson Laboratories, North Logan, UT) media containing 1% sucrose and 0.85% agar at 23° under conditions of 16 hr of light and 8 hr of darkness for 2 weeks. Wild-type (WT3w) and xrn2-1xrn3-3 were grown in soil (Metro Mix 250; Grace-Sierra, Boca Raton, FL) at 23° under conditions of 16 hr of light and 8 hr of darkness for 3 weeks. Total RNA was extracted from the plants using Trizol reagent (Invitrogen, Carlsbad, CA). Poly(A)+ fraction was separated from 80 μg of total RNA using Oligotex mRNA Mini Kit (Qiagen, Valencia, CA) and fragmented using Fragmentation Reagents (Applied Biosystems/Ambion, Austin, TX) at 70° for 15 min. Fifty nanograms of the fragmented RNA was used to generate strand-specific RNA-Seq library according to the Directional mRNA-Seq Library Preparation Protocol (Illumina). RNA-Seq libraries were sequenced for 42 cycles using the Illumina Genome Analyzer IIx (WT and fry1-6) or for 50 cycles using the HiSeq 2000 (xrn3-3, xrn3-3xrn4-6, 3-week-old WT and xrn2-1xrn3-3) according to manufacturer’s instruction. Image analysis and base calling were performed with the standard Illumina pipeline. Read sequences were aligned with the Tophat software to the TAIR9 reference genome (). Reads that aligned to multiple positions were discarded. Reads per kilobase of transcript per million (RPKM) values were calculated using the Refiner Genome module developed by Genedata Expressionist (Genedata Inc., Lexington, MA). The Arabidopsis genome annotation used was based on the TAIR9 (ftp://ftp.arabidopsis.org/home/tair/Genes/TAIR9_genome_release/). The transcripts predominantly upregulated in the mutants were identified by using a Student t-test (P-value < 0.1). […]

Pipeline specifications

Software tools TopHat, Genedata Expressionist
Databases TAIR
Application RNA-seq analysis
Organisms Arabidopsis thaliana, Saccharomyces cerevisiae