Superase in rnaase inhibitor

The Ribo-seq protocol was modified according to a previous study using 2.5 × 10⁶ cells or approximately 10 µg of RNA^{38 (link)}. After optimization, we lowered the input to 50 mouse oocytes. Fifty denuded mouse oocytes were lysed in Ribo-seq lysis buffer (1% Triton X-100, 20 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 100 µg/µL cycloheximide). The total DNA and RNA were digested by a mix of 1 U TURBO™ DNase (AM2239, Invitrogen), 0.02 U RNase A, and 0.8 U RNase T1 Cocktail (AM2286, Invitrogen) at 37 °C for 30 min. The digestion was terminated using SUPERase•In™ RNAase inhibitor (AM2696, Invitrogen). RNAs were purified using TRIzol (15596026, Invitrogen) in phasemaker tubes (A33248, Invitrogen), and the resulting RNAs were treated with T4 PNK (M0201S, NEB) to conduct end-repair. Ribosome-protected fragments (RPFs) were separated using 40% RNA PAGE-gel. Resulting 26‒40 nt bands were cut and minced using Squisher-Single (H1001, ZYMO) and soaked in recovery buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.25% v/v SDS, 10 mM MgCl₂) overnight. The RPFs were recovered by isopropanol precipitation. Libraries were then constructed using a SMARTer® smRNA-Seq Kit for Illumina (635030, TAKARA). Finally, samples were quantified using the Bioanalyzer High-Sensitivity DNA assay (Agilent Technologies) and sequenced using the Novaseq 6000 platform.

To demonstrate binding of the lncRNA to proteins, the RNA immunoprecipitation assay was used as described previously [17 (link)] with minor modifications.
Briefly, the nuclear fraction of cells was harvested using a nuclear isolation and lysis buffer (1.28 M sucrose, 40 mM Tris-HCl (pH 7.5), 20 mM MgCl₂ and 4% Triton X-100) followed by centrifugation. After resuspending in RIP buffer (150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/mL SUPERase In RNAase inhibitor (Invitrogen), 1 X cOmplete ULTRA (Roche)), chromatin was sheared by sonication. Dynabeads (Protein G, Lifetechnologies) were loaded with antibodies against p65 or cJUN by incubation at 4°C for three hours and then transferred to chromatin-sheared cell lysates for overnight incubation. After washing with RIP buffer, TRIzol was added to isolate RNA from RNA-protein complexes. Finally, reverse-transcription and qPCR were performed as above to amplify RNA that was bound to p65 or cJUN.