NextSeq 550 sequencing; Ribosome profiling and parallel RNA-seq of S. pombe cells subject to amino acid starvation
Ribosome profiling and parallel RNA-seq of S. pombe cells subject to amino acid starvation
Cells were incubated with CHX at 100 ug/ul for 5 minutes before collection Cells were grown at 32C in EMM2 (Moreno et al. Methods Enzymology 194: 795). 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).