Computational protocol: The Nuclear Exosome Is Active and Important during Budding Yeast Meiosis

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Protocol publication

[…] Cbc2 IPs were performed on 1.6×1010 cells using IP method 1 (see below), except that TEV elution was for 2 hours at 16° and calmodulin binding was for 50 min at 4°. To prepare RNAseq libraries, RNA was fragmented in 200 mM Tris acetate pH 8.2, 500 mM KOAc, 150 mM MgOAc2 for 3 min at 94°, ethanol precipitated and reverse transcribed from random hexamers using Superscript II (Life Technologies) according to manufacturer's instructions. Tris pH 7.8 and MgCl2 were added to 50 mM and 5 mM respectively, and second strand synthesis was performed with 2U RNase H (NEB) and 50U DNA polymerase I (NEB) at 16° for 2.5 hours. Ends were repaired using 15U T4 DNA polymerase (NEB), 5U Klenow DNA polymerase (NEB) and 50U T4 PNK (NEB) in T4 DNA ligase buffer (Life Technologies) at 20° for 30 min. cDNA was tailed with dATP using 15U Klenow (3′-5′ exo-) (NEB) in NEBuffer 2 at 37° for 30 min, and 1∶10 diluted Illumina adapters were ligated using a Ligafast kit (Promega) followed by gel purification of ∼200 bp species. Libraries were amplified for 15 cycles using Phusion polymerase (NEB) and Illumina PCR primers 1.0 and 2.0. Wild type and trf4Δ samples were sequenced on separate lanes of an Illumina GA-II system. Reads were mapped to the yeast reference genome (SGD1.01) or a custom assembled SK1 genome using Bowtie , allowing either unique mapping reads only or allowing non-unique reads to map at random respectively. For analysis, reads were summed in 100 bp segments spanning the genome using SeqMonk (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/), and reads from the 37S pre-rRNA were filtered out of the analysis as these represent an abundant contaminant of non-Cbc2-bound transcript. Total read-count normalisation was then applied to account for differing sequencing depths (16 million mapped reads for wild type, 21 million for trf4Δ). Analyses were performed using an R script (). Sequencing data is deposited at GEO, accession number GSE60221. […]

Pipeline specifications

Software tools Bowtie, SeqMonk
Application RNA-seq analysis
Organisms Saccharomyces cerevisiae