title: GSM5512591: WT_total_A; Schizosaccharomyces pombe; RNA-Seq
library_construction_protocol: Briefly, ribosome protected fragments were generated by digesting 100 A260 units of cleared lysate with 600 U RNase I (ThermoFisher) at 22°C. The reaction was stopped after 1 h by addition of 15 µl Superase In (ThermoFisher) and loaded on a 10–50% (w/v) sucrose gradient in 50 mM Tris-HCl pH 7.5, 50 mM NH4Cl, 12 mM MgCl2, 0.5 mM DTT, and centrifuged for 4 h at 38 krpm and 4°C in a SW41Ti rotor. Monosomes were fractionated and total RNA isolated. Subsequently, RNA was separated on 15% polyacrylamide gels (8 M urea, 1× TBE buffer) and 28–32 nt ribosome footprints were extracted from the gel. Purified ribosome protected fragments were cloned with primers that contained four randomized nucleotides to reduce ligation biases and were prepared for strand-specific NGS with the TruSeq Stranded Total RNA Sample Preparation Protocol (Illumina).