Ribo-SN-rep2 (CRX2069176)
50 mouse oocytes (per sample) were lyzed in 150 ul of ice-cold lysis buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 1% triton X-100 and 1 mM DTT] for 5 min on ice followed by snap-freezing in liquid nitrogen and thawing at 37oC three times. After spinning at 21300 xg for 5 min at 4oC, the supernatant was digested with 0.25 ul of 2 U/ul TURBO DNase (Thermo Fisher Scientific) and 0.2 ul of 1:10-diluted 20 U/ul RNase T1/0.5 U/ul RNase A cocktail (Thermo Fisher Scientific) for 30 min at 37oC. 5 ul of 20 U/ul SUPERaseIn RNase inhibitor (Thermo Fisher Scientific) and 95 ul of ice-cold lysis buffer were further added, and the mixture was extracted using TRIzol LS Reagent in Phasemaker Tube. Purified RNAs were resuspended in 5 ul of PNK solution [5 U T4 PNK (NEB) in 1X T4 PNK reaction buffer] and incubated for 10 min at 37oC. After pre-incubation, ATP was added to a final concentration of 10 mM and the mixture was incubated for another 30 min at 37oC before adding 5.5 ul of Gel Loading Buffer II (Thermo Fisher Scientific). Ribosome-protected fragments (RPFs) corresponding to the 25 to 35 nt band were separated using 6% TBE-Urea Gel (Thermo Fisher Scientific) and subjected to library preparation using NEXTFLEX Small RNA Seq Kit v3 (Perkin Elmer). The purified libraries were quantified by Qubit 1X dsDNA HS Assay Kit and qPCR, analyzed by a Fragment Analyzer System to confirm successful amplification and quality, and sequenced using Novaseq 6000 Sequencing System.