Preparation of samples for Ribo-seq

1. We grew S. cerevisiae cells exponentially at 30 °C in two 1 L flasks containing 200 mL of YPD medium.
2. When the culture density reached A₆₀₀ ~ 0.6, the cells were treated with 1.5 mg/mL telithromycin (Comment: this step can be skipped depending on your experiment).
3. Treated cells were collected by rapid filtration through Express Plus® Membrane filter (Millipore). Cells were flesh frozen in liquid nitrogen (Comment: the cells can be stored in -80°C frige for at least several weeks untill the next step).
4. The cells were lysed using Mixer Mill MM400 (Retsch) with 300 µL of lysis buffer (10 mM Hepes-KOH, pH 7.5, 50 mM KOAc, 10 mM NH₄Cl, 2 mM DTT, 5 mM Mg(OAc)₂) without addition of cycloheximide (Comment: in our case cells were treated with telithromycin).
5. Lysates were cleared by centrifugation (10 min 20,000 × g at 4 °C) and supernatants were treated with 30 U/A₂₆₀ of RNaseI (Ambion) at 4 °C for 5 min (Comment: for each new RNaseI preparation I recommend titrating amount of the RNase and time of treatment and analyze conversion of polysomes into the 80S ribosomes by sucrose gradient).
6. Three hundred microliter of digested lysates were loaded onto 500 µL of 25% (w/v) sucrose cushion in buffer 20 mM Tris-HCl, pH 8.0, 140 mM KCl, 10 mM MgCl₂ supplemented with 1× Complete protease inhibitor. Ribosomes were pelleted by centrifugation in a TLA100.2 rotor at 90,000 rpm (350,000 g), 4 °C for 1 h.
7. Pellets were resuspended in 500 µL of 1% SDS and ribosome-protected mRNA fragments were isolated by standard phenol-chloroform extraction followed by ethanol precipitation.
8. RNA pellets were resuspended in 20 µL of TE buffer and half of it was run in 15% polyacrylamide denaturing gel (Comment: another 10 µL of the isolated RNA can be stored at -80°C).
9. RNA footprints ranging in size between ~20 and ~35 nt were excised and eluted from the gel.
10. RNA footprints were converted to sequencing libraries as described by Becker et al.⁸⁹ (for replicate 1) or McGlincy & Ingolia⁸⁸ (for replicate 2). (Comment: I recommend using McGlincy & Ingolia protocol (Methods, 126, 112-129 (2017)).
11. The libraries were sequenced at the NUSeq Core (Northwestern University) on Illumina HiSeq 4000 platform.

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