GSM5742619: Y2; Homo sapiens; RNA-Seq
For regular RNA seq, Kidneys were snap-frozen and RNA was extracted using Zymo Direct-zol RNA kit. For ribo-seq, polysome buffer (1 mL per one mouse kidney) consisted of 20 mM Tris pH 7.5 (140 µL of 1 M Tris 7.0 plus 60 µL of 1 M Tris 8.0 for 10 mL final buffer), 100 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 0.1% sodium deoxycholate, DTT, 0.02 U/µL Turbo DNAse (ThermoFisher AM2238), 200 µg/mL cycloheximide, and Protease Inhibitor Cocktail Tablet. for regular RNA seq, cDNA library preparation involved mRNA capture/enrichment, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification, following the KAPA mRNA Hyper Prep Kit Technical Data Sheet, KR1352 – v4.17 (Roche). For ribo-seq, RNase I digested, size selected RNA fragments were treated with T4 PNK and adapter ligation was done using NEXTFLEX Small RNA-seq kit v3 (PerkinElmer NOVA-5132-05).