GSM5664660: KO3_YAMAT; Mus musculus; OTHER
5ug total RNA was deacetylated to remove amino acids from 3' ends and demethylated to remove m1A, m1C, and m3C modifications using a proprietary demethylation mix (Arraystar, Inc).  40um of YAMAT forked linkers (Y-3-AD_UMI: 5'-P-GTATCCAGTNNNNTGGAATTCTCGGGTGCCAAGG-3'-ddC; Y-5-AD_UMI: 5'-GTTCAGAGTTCTACAGTCCGACGATCNNNNACTGGATACTGrGrN-3') were then incubated with pure demethylated and deacetylated RNA followed by addition of 10X annealing buffer (50 mM Tris HCl pH 8, 100 mM MgCl2, 5 mM EDTA) and then overnight incubation with T4 RNA ligase 2.  Linker-ligated RNA was then incubated with RT Primer (TruSeq Small RNA Library Prep Kit, Illumina) and reverse transcription was performed with Superscript III RT (Invitrogen) followed by bead purification.  Libraries were amplified using Phusion Hotstart II Polymerase (Thermo Scientific) with primers and indexes from the TruSeq Small RNA kit (Illumina) for 11 PCR cycles.  Amplified libraries were then bead-purified and run on the Agilent Bioanalyzer for confirmation of library size and quantified with the Qubit Fluorometer (Invitrogen). total RNA was extracted from mouse cortex using Trizol extraction and quantity was measured on Qubit fluorometer.  Quality of total RNA was checked on the Agilent Bioanalyzer using the RNA 6000 Pico Kit