GSM5661158: Upf1FLAG_upf2Δ_CHX_IP  rep1_riboseq; Saccharomyces cerevisiae; OTHER
Cells were lysed in a cryomill (Retsch).  Frozen powder from a given sample was pooled, thawed and clarified by ultracentrifugation. For ribosome profiling, lysates were digested with RNaseI and ribosomes were isolated over 1 M sucrose cushion.  For immunopurified samples, ribosomes were subsequently immunopurified over α-FLAG M2 affinity gel (Sigma, #A2220). RNAs were isolated from ribosomes (for ribosome profiling) or from lysates (for RNA-seq) using the miRNeasy kit (QIAgen, #217004). For ribosome profiling, ribosomal RNAs were depleted using the Yeast Ribo-Zero Gold rRNA removal kit (Illumina, #MRZY1324) or QIAseq FastSelect –rRNA Yeast Kit (#334215) and sequencing library was prepared using the NEXTFlex Small RNA-Seq Kit v3 (BIOO Scientific, #NOVA-5132-06). For RNA-seq, total RNAs were treated with BaselineZero DNase, depleted of rRNAs using the Yeast Ribo-Zero Gold rRNA removal kit (Illumina, #MRZY1324) or QIAseq FastSelect –rRNA Yeast Kit (#334215), then the sequencing library was prepared using the TruSeq Stranded mRNA sample prep kit (Illumina, #RS-122-2101)