title: GSM4648155: Fly WT Male 1-3d tRNA BS; Drosophila melanogaster; Bisulfite-Seq
library_construction_protocol: 20 ug total RNA was first loaded into 15% denaturing TBE-Urea PAGE Gels and separated based on the molecular weight. The gel slice corresponding to 70-90 nucleotides was excised from the PAGE gel and the RNA fragments were recovered using ZR small-RNA™ PAGE Recovery Kit (Zymo Research) following the manufacturer's protocol. 100ng-1μg of RNA was converted using the EZ RNA methylation kit (Zymo Research) with 3 cycles of [70 C 10 min, 64 C 45 min] and 37 C 30 min for desulfonation. End-repair was performed with T4 PNK (NEB) to phosphorylate 5'-hydroxyl termini and remove 2',3'-Cyclic phosphate produced during the bisulfite/desulfonation reaction. The tRNA BS-seq library was generated using VAHTSTM Small RNA Library Prep Kit for Illumina (Vazyme) according to the manufacturer's protocol.