Illumina HiSeq 2500 sequencing; Ribosome profiling of NEW1 knock-out of budding yeast Saccharomyces cerevisiae
Ribosome profiling of NEW1 knock-out of budding yeast Saccharomyces cerevisiae
Cells were collected by rapid vacuum filtration and subsequent flash-freezing in liquid nitrogen. Frozen cell pellets were lysed using cryogenic milling. Cultured at 20 degrees Celsius in 750 ml of synthetic complete medium up to OD600 of 0.6. 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: genotype
wild type genotype
Experimental Factor: protocol
Ribo-Seq