Illumina HiSeq 2000 sequencing; Effects of cycloheximide (CHX) on ribosome profiling experiments in Schizosaccharomyces pombe
Effects of cycloheximide (CHX) on ribosome profiling experiments in Schizosaccharomyces pombe
wt.N.CHX.mrna.1_s
Cells were grown at a temperature of 32C in EMM2 (Moreno et al. Methods Enzymology 194: 795) containing NH4Cl as a nitrogen source Cells were grown in the presence of CHX at 100 ug/ml for 5 minutes before colletion Total RNA was extracted using a hot phenol extraction method as described in Lyne et al. (2003) BMC Genomics 4:27. Clean up and DNase treatment were performed on with Purelink RNA microcolumns (Life Technologies) following the manufacturer’s instructions.  Samples were ribo-depleted using Ribo-Zero Gold rRNA Removal Kit Yeast (Illumina) with a 4 ug input.   30 ng of ribo-depleted RNA was used as starting material for the NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific).