Illumina HiSeq 2500 sequencing; Ribosome profiling of budding yeast Saccharomyces cerevisiae in eEF3 depleted conditions
Ribosome profiling of budding yeast Saccharomyces cerevisiae in eEF3 depleted conditions
Cells were collected by rapid vacuum filtration and subsequent flash-freezing in liquid nitrogen and cryogenic milling. Cultured at 30 degrees Celsius in 700 ml of synthetic drop-out SC -met-cys medium supplemented with or without 0.5 mM of methionine. TRI Reagent (AM9738, ThermoFisher) was used to extract the RNA, followed by clean-up using RNA Clean & Concentrator -25 Kit (Zymo Research).  RNA-Seq libraries were constructed using ScriptSeq Complete Gold Yeast Kit (Epicentre). Ribo-Seq libraries were prepared essentially as per Ingolia and colleagues, NatMeth 2012 (PMID: 22836135) with modifications on rRNA removal (Ribo-Zero Gold rRNA Removal Kit -Yeast) and sample purification procedures between enzymatic steps (RNA Clean & Concentrator -5 Kit).
Experimental Factor: compound
none
Experimental Factor: fraction
rRNA-depleted total RNA