GSM3533053: Pooled RPF; Saccharomyces cerevisiae; OTHER
Cells were grown overnight in rich media and subsequently shifted to minimal media till the OD600 reaches 0.5-0.8. Cells were rapidly harvested by filtration and lysed, as described in detail (Weinberg et al., 2016; McGlincy and Ingolia, 2017). For both transcriptome and ribosome profiling analyses, three biological replicates each for WT and tRNA thiolation mutant cells (∆uba4 and ∆ncs2) were included. Separate 5' and 3' linkers were ligated to the RNA-fragment instead of 3' linker followed by circularization (Subtelny et al, Nature 2014). 5' linkers contained 4 random nt unique molecular identifier (UMI) similar to a 5 nt UMI in 3' linkers. During size-selection, we restricted the footprint lengths to 18-34 nts. Matched RNA-seq libraries were prepared using RNA that was randomly fragmentation by incubating for 14 min at 95C with in 1 mM EDTA, 6 mM Na2CO3, 44 mM NaHCO3, pH 9.3. RNA-seq fragments were restricted to 18-50 nts. Ribosomal rRNA were removed from pooled RNA-seq and footprinting samples using RiboZero (Epicenter MRZY1324). cDNA for the pooled library were PCR amplified for 16 cycles.
GEO Accession
GSM3533053