GSM2793657: Ribo_noCHX_2_elp3D; Saccharomyces cerevisiae; OTHER
For CHX-free samples, cells were harvested by rapid filtration system and lysed with frozen pellets of lysis buffer using CryoMill in liquid nitrogen, followed by RNase I treatment. Ribosomes were separated on 10-50% sucrose gradient and 80S monosome fractions were collected and RNA extracted. 27-34 nt ribosome footprints were isolated by denaturing PAGE. RPF are constructed into deep-sequencing libraries as described in (Heyer et al., 2015). Briefly, RNA 3’ ends were dephosphorylated, ligated with a preadenylated adaptor, and reverse transcribed. cDNA was precipitated and size selected by denaturing PAGE. The purified cDNA was circularized and PCR amplified prior to sequencing. For RiboMeth-seq, total RNAs were end-repaired with T4 PNK, Antarctic phosphatase, or Shrimp alkaline phosphatase for 3’-end dephosphorylation and with T4 PNK and ATP for 5’-end phosphorylation, followed by library construction using NEB Next Small RNA Library Prep kit. Amplified library was purified and subject to Illumina Nextseq 500 single-end sequencing with 75 cycles.
GEO Accession
GSM2793657