GSM5412805: tRNA_MsLV_M_Expr3; Mus musculus; OTHER
Total RNA from approximately 30 mg of liver tissue powder was isolated using TRIzol and Phasemaker tubes according to the manufacturer's instructions. After RNA oxidation with 50 mM NaIO4 and β-elimination, two synthetic RNA standards were spiked-in followed by dephosphorylation. Mature tRNAs were purified by denaturing 10% TBE-Urea PAGE followed by ligation with the I4 3' adapter using T4 RNA ligase 2, truncated KQ. TGIRT was used for primer-dependent reverse transcription at 42˚C for 16 hours. Ethanol purified cDNA was circularized with CircLigase ssDNA ligase followed by PCR amplification. Libraries were purified with 8% PAGE and  quantiﬁed with the high sensitivity fragment analysis assay, pooled in equimolar ratios and sequenced for 150 cycles on NextSeq 500. mim-tRNA sequencing