GSM7899187: Ribo-seq, Beige Ad. Diff. 0d, rep2; Mus musculus; OTHER
Cultured cells were washed two times with 1X Phosphate-base buffer (PBS) and then incubated for 30 min on ice with the lysis buffer (10 mM/mL Tris-HCl, pH 7.4 (AM9850G, AM9855G, Invitrogen); 5 mM/mL MgCl2 (#AM9530G, Invitrogen); 100 mM/mL KCl (#AM9640G, Invitrogen); 2 mM/mL dithiothreitol (DTT, #707265ML, Thermo Fisher); 100 μg/mL cycloheximide (CHX, #C1988, Sigma‒Aldrich); 1% Triton X-100 (#T8787, Sigma‒Aldrich); 1X protease inhibitor (#539134, Sigma-Aldrich); 2 μL/mL SUPERase inhibitor (#AM2696, Invitrogen); and 2 μL/mL RNase inhibitor (#AM2694, Invitrogen). After centrifuging the lysates at 20000 g for 15 min at 4 °C, the supernatant was separated into three portions for RNA-seq, Ribo-Seq, and Mass Spectrometry. For Ribo-seq, the supernatant was treated with 3 U/mL RNase I (#EN0601, Thermo Fisher) for 30 min at 25 °C to digest mRNA regions not associated with ribosomes. The samples were passed through MicroSpin S400 HR Columns (#27-5140-01, Cytiva), and prewashed with a polysome buffer (20 mM HEPES-KOH, pH 7.4 (#H0887, Sigma‒Aldrich); 5 mM MgCl2; 100 mM KCl; 2 mM DTT; and 100 μg/mL CHX). The filtered samples of ribosome-protected mRNA-containing lysate excluding the digested mRNA fragments were treated with TRIzol LS to isolate RNA. Ribosomal RNAs were removed using a Ribo-Zero Gold Kit of the TruSeq Stranded Total RNA Library Prep Gold protocol (#20020599 Illumina). The RNA samples were then precipitated in 100% ethanol (#A995-1, Thermo Fisher) at -20 °C and labeled with the radioisotope γ-P32 ATP (#NEG-502H-1 PerkinElmer) using T4 Polynucleotide kinase (T4 PNK, #M0201L New England Biolabs) and separated on a 10% denaturing Urea-PAGE (#U5128, Sigma‒Aldrich) gel. The signals were visualized using an Amersham Typhoon Biomolecular imager (v2.0.0.6 GE Healthcare). Following the analysis of radioisotope signals with MultiGauge (v3.0) software, the gel was excised to isolate RNA samples of approximately 30 nucleotides. The extracted RNA from a gel with RNA elution buffer (0.3M NaOAc, #AM9740, Invitrogen; 2% sodium dodecyl sulfate) was then dephosphorylated using Antarctic Phosphatase (#M0289S New England Biolabs) and relabeled with γ-P32 ATP using the T4 PNK reaction. The labeled samples were separated on a 10% denaturing Urea-PAGE gel to remove free ATPs, followed by gel excision and RNA extraction. Adapter ligation and PCR amplification were performed using a RealSeq®-AC miRNA Library Kit (#500-00048, RealSeq Biosciences). The adapter-ligated PCR products were purified with SPRIselect magnetic beads and then subjected to sequencing in the NovaSeq 6000 system.