GSM5114107: AHARIBO RIBO-Seq from mESCs, replicate 2; Mus musculus; OTHER
Standard RIBO-seq. 80S ribosomes were isolated by centrifuging lysates through a 30% sucrose standard at 95000 rpm, for 2 h at 4°C. The standard was resuspended in W-buffer and treated with SDS 10% (final 1%) and 5 µL of proteinase K (20mg/mL), and incubated at 37 °C in a water bath for 75 min before acid phenol:chlorophorm:isoamyl alcohol (pH 4.5) RNA extraction.  AHARIBO RIBO-seq. The lysates was incubated with Dibenzocyclooctyne-PEG4-biotin conjugate (Sigma-Aldrich #760749; 50 µM final concentration) in a reaction volume of 100 µl for 1 h on a rotator in slow motion (9 rpm) at 4°C. Lysates were then incubated with 50 µl of eMagSi-cN beads (IMMAGINA BioTechnology #018-eMS-001) for 30 min at 4°C on the rotator in slow motion (9 rpm). Subsequently, samples were taken off the rotator and placed on a magnetic rack on ice, and supernatants were discarded. Beads were washed two times with 500 µl of 1x PBS supplemented with 0.1 % Triton-X100, 1x sBlock and 1:10,000 RiboLock RNase Inhibitor (Thermo Scientific #EO0381) on the rotator in slow motion at 4 °C, removing supernatants from the tubes sitting on the magnetic rack and gently adding new washing solution each time. After the final wash, beads were resuspended in 200 µl of W-buffer and transferred to a new vial. Then, 20 µl of 10% SDS and 5 ul of Proteinase K (Qiagen #19131) were added to each sample, and samples were incubated at 37 °C for 75 min in a water bath. Subsequently, suspensions were transferred to a new vial and acid phenol:chloroform:isoamyl alcohol (pH 4.5) RNA extraction was performed. Extracted RNA was then resolved by electrophoresis through denaturing TBE-urea gels, and fragments between 25 nt and 35 nt were excised. Libraries were prepared using the RiboLace kit_module 2 (IMMAGINA BioTechnolgy #RL001_mod2) and sequenced on an Illumina HiSeq 2500 sequencer with a single-end 50 base pair run.