GSM5239804: cyt-3: cyt DHFR - without CHX - Replicate 3; Saccharomyces cerevisiae; OTHER
Cells were harvested by fast filtration, flash-frozen in liquid nitrogen and lysed in a Retsch bead mill under cryogenic conditions. Cell powder was thawed and footprints were generated by RNase I digestion. Ribosomes were pelleted through a 25% (w/v) sucrose cushion by centrifugation for 20 min at 72,000 rpm in a Beckman TLA 100.2 rotor. RNA was extracted with hot acid phenol:chloroform. Ribosome footprints were size selected by TBE-Urea PAGE. rRNA was removed with the Ribo-zero Gold kit (Illumina). RNA footprints were dephosphorylated, ligated to a linker sequence, reverse-transcribed to cDNA and circularized. Illumina sequencing barcodes were added by PCR amplification. Libraries were quantified with qPCR (Kapa Biosystems)