title: GSM6153410: WT heart RNA-seq replicate 3; Mus musculus; RNA-Seq
library_construction_protocol: C2C12 cells were lysed with a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, and EDTA-free protease inhibitor cocktail] supplemented with cycloheximide (100 μg/ml). 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. 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).