GSM1646016: WT_ribo_YPD_rep2; Saccharomyces cerevisiae; RNA-Seq
Frozen cells were pulverized under cryogenic conditions in 20 mM Tris-HCl (pH=7.5), 100 mM NaCl, 10 mM MgCl2, 1% Triton X100, 0.5 mM DTT, 100 µg/ml CHX. Extracts were treated with RNAse I and resolved on sucrose gradients. RNA from monosome fractions was extracted by hot phenol and ribosome footprints of 28-32 nt were purified by size selection and gel extraction. Footprints were dephosphorylated and ligated to a preadenylated 3' linker. Following reverse transcription, cDNA was circularized and amplified by PCR. Footprints were dephosphorylated and ligated to a preadenylated 3' linker. Following reverse transcription, cDNA was circularized and amplified by PCR.