Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Cells were lysed on ice in the presence of cycloheximide. Lysates were clarified by centrifugation. For ribosome profiling, monosomes were generated by RNaseI digest followed by ultracentrifugation. For RNAseq, total RNA was extracted using Trizol, and sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep Gold (20020598) kit. For ribosome profiling, fragments ranging from 15-34 nt were gel purified. Upon dephosphorylation of sample RNA, linker was ligated and this product and and rRNA was depleted using Ribo-Zero Gold from Illumina. This was followed by reverse transcription and gel purification. Libraries were then circularized, PCR-amplified, and gel-purified cDNA was submitted for sequencing. RNA-seq samples were prepared from 1 ug of total RNA following the Truseq protocol.