GSM9270426: DLD-1 cells, replicate 3, RanGAP1 KD, hour 5, 1 hour 4sU labeling; Homo sapiens; OTHER
Total RNA harvested in TRIzol reagent in DNA LoBind tubes. 10% 4sU-labeled and unlabeled ERCC spike-ins added proportional to harvested cell number. RNA extracted with Zymo RNA Clean & Concentrate columns. 3ug total RNA was taken into ribosomal RNA depletion (Z. Zhang et al., 2012). rRNA-depleted RNA was treated with 10mM iodoacetamide to alkylate 4sU bases (Herzog et al., 2017). 100ng rRNA-depleted, IAA-treated RNA per sample was used as input to NEBNext Ultra II directional RNA library prep kit and libraries were prepared according to manufacturer's recommendations. Library quality was assessed on Agilent TAPE station.