Ribo-Seq of yeast W303: WT
Whole cell lysates were treated with RNaseI (Invitrogen) and ultracentrifuged through a sucrose cusion to isolate 80S ribosomes. Ribosome-protected mRNA fragments (RPFs) were isloated from 80S ribosomes using miRNeasy kit (QIAGEN), size selected for RNAs < 200 nt, and 3'-dephosphorylated/5'phosphorylated by T4 polynucleotide kinase (NEB). rRNAs were removed using QIAseq FastSelect - rRNA Yeast kit (QIAGEN) and sequencing libraries were prepared using NEXTFLEX Small RNA-Seq Kit v3 (Perkin Elmer), skipping the first denaturation step.