Saccharomyces paradoxus Ribosome profiling mRNA fraction
Ribosome profiling next-generation sequencing (NGS) libraries were prepared as detailed in [Ingolia NT. 2010. Genome-Wide Translational Profiling by Ribosome Footprinting. In Methods in Enzymology Vol. 470 (eds. J Weissman, C Guthrie, GR Fink), pp. 119–142. Academic Press, Waltham, Massachusetts] with the following modifications: Cryo-grinding of lysates was performed in a Retsch Mixer-Mill MM 301 (Retsch Technology GmbH) at maximum frequency for two 1.5 minute cycles with immersion in liquid N2 before grinding, in between cycles, and following grinding. After purification of the cryo-ground lysate, RNA abundance was determined from the A260 measured using a Nanodrop 2000c (Thermo Scientific) and 1000 µg of RNA was subjected to density gradient centrifugation for monosome isolation. Gradients were fractionated and fractions corresponding to the 80S monosome were collected using a Biocomp Instruments Gradient Station attached to a Foxy Jr Fraction Collector (Teledyne Isco). Total RNA was isolated from the purified lysate using the Epicenter MasterPure™ Yeast RNA Purification Kit beginning with 500 µg of lysate RNA diluted to 125 µl using polysome lysate buffer. Following circularization, the libraries were subjected to an rRNA subtraction step as described in [Brar GA, et al. 2012. High-resolution view of the yeast meiotic program revealed by ribosome profiling. Science 335: 552–557].