title: Ribosome profiling of Saccharomyces cerevisiae BY4147 30min at 37C
design: The RNA was isolated from logarithmic growing, heat-stressed yeast cells (37 C, 30 min) according to the TruSeq RiboProfile Kit (yeast; Illumina). For RNA footprinting 15 U/A260 of 100 U/l RNase I (Ambion) were added to 200 l of the cell lysate. The reactions were incubated at RT and 350 rpm. After 1 h the reactions were stopped by the addition of 10 l 20 U/l SUPERaseIn RNase Inhibitor (Illumina) and the samples chilled on ice until separation via MicroSpin S-400 columns. After purification of the ribosome protected fragments (RPFs), rRNA was depleted from the RPF according to the Ribo-Zero Magnetic Gold Kit (Illumina) procedure. Subsequently, the RPFs were gel-purified with 15 % TBE/Urea gels (life technologies) and excised from the gel. For RNA extraction the gel slices were transferred into a 0.5 ml Eppendorf tube with holes punched into the bottom and centrifuged into 1.5 ml Eppendorf tubes (ca. 5 min at 12000 x g). The shredded gel slices were suspended in 442 l 0.45 M ammonium acetate, 0.045 % SDS solution and rocked at RT for 3-4 h. Then the gel was removed by filtration over centrifugal filters (0.22 m, Merck Millipore), 700 l of 100 % Isopropanol and 2 l Glycogen solution (Illumina) were added and the RNA precipitated at -20 C overnight. End repair, 3 adapter ligation, reverse transcription cDNA circularization and PCR were performed as described in the TruSeq RiboProfile Kit. The libraries were sequenced on a HiSeq1500 sequencer (Illumina).