title: GSM6924851: WT m6A-seq 2; Mus musculus; RIP-Seq
library_construction_protocol: The heart tissues were flash frozen in liquid nitrogen and lysed in a lysis buffer with the use of Multi beads shocker (YASUI KIKAI). The lysates were centrifuged at 20,380 × g for 10 min at 4°C, and the resulting supernatants were collected, assayed for RNA concentration with a Qubit RNA BR Assay Kit (Thermo Fisher Scientific), incubated with RNase I (20 U per 10 μg of RNA, Epicentre) for 45 min at 25°C, and placed on ice before the addition of SUPERase•In RNase inhibitor (Invitrogen). The samples were then loaded on a 1 M sucrose cushion [containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, SUPERase•In (10 U/ml), 1 mM dithiothreitol, and cycloheximide (100 μg/ml)], and the gradients were centrifuged at 100,000 rpm (417,200 × g) for 1 h at 4°C in a Beckman TLA110 rotor. The resulting pellets were suspended in ribosome splitting buffer [20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, SUPERase•In (20 U/ml), 1 mM dithiothreitol, and 1% Triton X-100] and subjected to purification with the use of an Amicon Ultra filtration device (100-kDa cutoff, Millipore) to deplete rRNAs. Total RNA was extracted from tissue lysates with the use of a PureLink RNA Mini Kit (Thermo Fisher Scientific), and the quality of the purified RNA was assessed with a 2100 Bioanalyzer (Agilent). The purified RNA was subjected to selection on the basis of a size range of 17 to 34 nt for Ribo-seq and 50 to 80 nt for Disome-seq by electrophoresis through a 15% polyacrylamide and Tris-borate-EDTA (TBE)–urea gel (SuperSep RNA, Fujifilm). The footprint fragments were treated with T4 polynucleotide kinase (New England Biolabs) to repair the 2'-3' cyclic phosphates, a DNA linker including barcode sequences (NI-810 to NI-817) was ligated with the use of T4 RNA ligase 2, truncated K227Q (New England Biolabs), and the resulting products were purified on a 15% polyacrylamide and TBE-urea gel. Ribosomal RNAs were further depleted with the use of RiboZero Gold (Illumina). Reverse transcription (RT) was performed with the NI-802 primer, and the resulting products were purified on a 15% polyacrylamide and TBE-urea gel. The purified cDNAs were circularized with circLigase II (Lucigen), and index sequences were then added by amplification with the polymerase chain reaction (PCR) as performed with the common primer (NI-798) and primers including index sequences (NI-799 and NI-822 to NI-826). Products of the desired size were purified on a 15% polyacrylamide nondenaturing gel (SuperSep DNA, Fujifilm), and the libraries were sequenced with a NovaSeq 6000 system (Illumina). tRNA-seq library construction protocol Periodic oxidation of 10   g of total RNA extracted from mouse heart was carried out in 100 mM NaOAc/HOAc (pH 4.8) and 50 mM NaIO4 for 30 min at room temperature. Then, 1 M glucose was added to a final concentration of 100 mM, left at room temperature for 5 minutes, and RNA was purified using Micro Bio-Spin P-6 Columns (Bio-Rad). The RNA was treated with 60 mM sodium borate (pH 9.5) at 45  C for 90 min for deacylation, and purified again with Micro Bio-Spin P-6 Columns. Purified RNA was selected according to size in the range of 70 to 90 nt by electrophoresis in a 15% polyacrylamide and TBE-urea gel, and demethylated using demethylase supplied with the rtStarTM tRNA-optimized First-Strand cDNA Synthesis Kit (Filgen). The resulting sample was then subjected to dephosphorylation, DNA linker ligation, and rRNA depletion with RiboZero Gold as described for the Ribo-seq protocol. RT was performed with the use of the SI-019 primer, and the resulting products were purified on a 15% polyacrylamide and TBE-urea gel. The second linker (SI-018) was ligated by incubation overnight at 25  C with T4 RNA Ligase 1 (ssRNA Ligase), High Concentration (New England Biolabs). The products were then amplified by PCR with the same primers as used for the Ribo-seq protocol, and those of the desired size were purified by electrophoresis through a 15% polyacrylamide nondenaturing gel. m6A-seq library construction protocol m6A-seq libraries were prepared with the use of EpiNext CUT&RUN RNA m6A-Seq Kit (EpiGentek) according to the manufacturer's protocol. Briefly, 5   g of total RNA extracted from mouse heart was immunoprecipitated with m6A antibody and cleaved on beads. The beads were then washed, RNA was purified from the beads, reverse transcribed, and the resulting cDNA was amplified by PCR.