title: GSM7062945: SUB280 rad6Δ+peroxide_rep1 [SM066M]; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: Cells were rapidly vacuum filtered and flash frozen. Ribosome profiling samples were lysed in a freezer mill with lysis buffer. Total RNA for mRNA-Seq was extracted from whole cells. Ribosome profiling lysates were clarified, treated with RNase I, and run over sucrose gradients to extract either the monosome or the disome peak. RNA was then extracted using SDS/hot phenol/chloroform and footprints of the specified size were retained by gel purification. Briefly, RNAs were ligated to a universal DNA linker followed by reverse transcription. cDNAs were then circularized and PCR amplified. Ribosome profiling circularized cDNA was subject to ribosomal RNA subtraction using custom biotinylated oligos in some cases. In others, Ribo-Zero was used. RNA-Seq samples were prepared with the Illumina Truseq Stranded ribo-zero kit.