GSM3665263: smallRNA-K63464-Liver-unoxidized; Phascolarctos cinereus; ncRNA-Seq
Total RNA samples were treated with Turbo DNase (Invitrogen) and RNA cleanup was done following manufacturer's instructions using the RNeasy Mini Kit (Qiagen). Small RNA sequencing libraries were prepared as previously described (Li, C., Vagin, V.V., Lee, S., Xu, J., Ma, S., Xi, H., Seitz, H., Horwich, M.D., Syrzycka, M., Honda, B.M., et al. (2009). Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. Cell 137, 509–521.). Briefly, total RNA was isolated from flash frozen koala tissue using the mirVana miRNA Isolation Kit (Ambion/Life Technologies). Small RNAs were sized selected and purified from a 15% denaturing polyacrylamide-urea gel using the ZR small-RNA™ PAGE Recovery Kit (Zymo Research). For oxidized RNA library preparations, purified RNA was oxidized with 25 mM NaIO4 in 30 mM borax, 30 mM boric acid, pH 8.6, for 30 min at room temperature followed by ethanol precipitation. 3′ pre-adenylated adapter was ligated to oxidized or un-oxidized small RNAs. The 3´ ligated product was purified from a 15% denaturing polyacrylamide-urea gel. 5′ RNA adapter ligation was performed and the ligated product was purified from a 10% denaturing polyacrylamide-urea gel and used to synthesize cDNA. The resulting cDNA was PCR amplified and run on a 2% Certified Low Range Ultra Agarose (Bio-Rad) gel with subsequent extraction using the QIAquick® Gel Extraction Kit (Qiagen).