Computational protocol: TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

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Protocol publication

[…] Adapter sequence (5′-AGATCGGAAGAGCACACGTCT-3′) was trimmed from raw reads using Cutadapt (). To reduce contamination from noncoding RNAs (such as rRNAs, tRNAs, small nuclear RNAs, and small nucleolar RNAs), trimmed reads were aligned to relevant sequences downloaded from the Saccharomyces Genome Database (; (downloads .yeastgenome.org/sequence/S288C_reference/rna/rna_coding .fasta.gz), using the Bowtie short read alignment program (), and no more than three mismatches were allowed for these alignments (-v 3). Reads aligning to these sequences were removed from the data set for further analysis. Read-length distributions were analyzed to determine the most prevalent read lengths among the samples. Reads of <20 or >80 nt were discarded from further analyses.The S. cerevisiae genome was obtained from the National Center for Biotechnology Information (GCA_000146055.2, dated December 2013; ), to which reads were aligned using Bowtie (). Parameters used for Bowtie alignments were -m 1 -v 3, that is, no more than three mismatches were allowed, and nonunique mappings were discarded. To evaluate the number of reads mapped on each gene, gene annotations were procured from the Saccharomyces Genome Database (). Reproducibility of the data from all the replicates was evaluated by calculating Pearson’s r. Specifically, the number of raw reads mapped on each gene for ribosomal profiling (two replicates) and total RNA-seq (three replicates) were used for calculating a Pearson’s r. […]

Pipeline specifications

Software tools cutadapt, Bowtie
Databases SGD
Application RNA-seq analysis
Organisms Saccharomyces cerevisiae