GSM4671040: L_SPF_UT_2; Mus musculus; OTHER
Experiments were carried out according to previously published protocols for DM-tRNA-seq (Zheng et al. Nat Methods. 2015;12:835–7). Dissected tissues were homogenized with glass beads using tissue homogenizer and total RNAs of them were isolated using TRIzol reagent (Ambion) following the manufacturer's protocol. RNA samples were dissolved in RNase-free water, and the concentration and purity of total RNAs were spectrophotometrically measured at 260 nm using a NanoDrop 2000 (Thermo Scientific). Total RNAs were firstly deacylated with Tris-HCl (pH = 9.0) at 37°C for 30 mins and then gel purified using 10% urea denaturing gel as standard procedures. The tRNA bands from each sample were cut off under the UV shadowing, and isolated total tRNAs (approximately 3 μg, 60 pmol) were dissolved in RNase-free water. About 1.5 μg total tRNA from each sample was treated with a 3× molar ratio of wild-type AlkB (180 pmol) and a 4× molar ratio of D135S mutant (240 pmol) to remove Watson–Crick face methylations on tRNAs that can impede the processivity of reverse transcription while another 1.5 μg total tRNA extracted from the same sample was untreated. Afterward, we performed reverse transcription of 150 ng of demethylase-treated (+DM) and untreated (−DM) tRNA with a thermophilic reverse transcriptase (TGIRT, InGex). We purified and eluded cDNAs on a 10% urea denaturing gel by using linear acrylamide (AM9520, Thermo Scientific) as the carrier. The purified cDNAs were circularized with CircLigase II (Epicentre) at 60°C overnight following the manufacturer's protocol and then extracted with phenol-chloroform-isoamyl alcohol (25: 24: 1) followed by ethanol precipitation For library construction, the circularized cDNAs were amplified with Phusion-HF (NEB) by PCR amplification of 12 cycles using Illumina multiplex (5'–AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC–3') and barcode (5'–CAAGCAGAAGACGGCATACGAGAT [Index] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT–3'). The PCR products were purified with AMPure XP beads (Beckman Coulter) and sequenced on an Illumina HiSeq system
GEO Accession
GSM4671040