GSM5610144: ribo_gir2_4_b1_s2; Saccharomyces cerevisiae; RNA-Seq
Cell lysates were clarified by two sequential centrifugation steps - 3000g, 5 minutes, 4℃, and 20000g, 10 minutes, 4℃. Part of the cell lysate was used for mRNA isolation using Oligo(dT) beads.  Another part was treated with ribonuclease I for polysome disassembly and applied to a linear 10-50% sucrose gradient in fractionation buffer (20 mM Tris pH 8.0, 140 mM KCl, 15 mM MgCl2, 1 mM DTT, 0.1 mg/ml cycloheximide, 1% Triton) and separated on a SW-41 rotor (Beckman) at 35000 rpm, 3 hours, 4℃. Subsequently, ribosome-bound RNA fragments were collected from the monosome fraction. Ribosome-bound RNA was isolated using acidic-phenol extraction. cDNA libraries for Ribo-Seq and RNA-Seq were constructed in parallel using standard ribosomal profiling library construction protocol (described in Kasari et al., 2019 DOI: 10.1093/nar/gkz600). Importantly, cycloheximide was added to the lysis buffer but not to the growth media.