title: "RATE-seq,Nitrogen-limited, medium growth rate, repeat 2, time 50 min"
design: First strand synthesis of rRNA-depleted RNA was performed using the Super Script III kit (Life Technologies) and random priming using random hexamers. Second-strand synthesis was performed with dUTP in place of dTTP to enable strand-specific sequencing (Parkhomchuk et al. 2009). Samples were end repaired, A-tailed, and ligated to NEXTflex DNA Barcodes for multiplex sequencing. Adapter dimers were removed using AMPure beads (Agencourt). Samples were then treated with UNG and amplified using 10 cycles of PCR prior to sequencing. Samples were sequenced using an Illumina 2000 single-end 50-bp run. Sequence alignment  Ilumina sequencing reads were first filtered for rRNA sequences by aligning to the ribosomal DNA of the yeast genome using Bowtie with default settings. All remaining reads were then aligned to the rest of the yeast genome and the three spike-in sequences using Bowtie2 (Langmead et al. 2009). After converting SAM files to BAM files, reads were filtered based on quality scores of 20 or higher. The resulting BAM files were then used to calculate total counts per transcript using the featureCounts function of the Subread package in R, using the argument for strand specific counting. Each library had between 5 and 13 million reads mapping to non-rRNA transcripts. 