GSM5219247: HeLa LENG8-input; Homo sapiens; RIP-Seq
HeLa cells expressing GFP-tagged human THOC1 or LENG8 were pelleted by centrifugation at 500g for 10 min at 4 °C and washed twice with ice-cold PBS. Cells were lysed in an equal volume of RIP lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 5 mM MgCl2, 25 mM EDTA, 0.5% (v/v) Nonidet-P40, 1 mM dithiothreitol, protease inhibitor cocktail (EDTA-free, Beyotime) for 30 min on ice in the presence of 100 U ml−1 RNase inhibitor (Sangon)) and lysates clarified by centrifugation at 9,000g and 4 °C for 10 min. Clarified lysates were incubated with anti-GFP with a final concentration at 0.2ug ml-1 and the binding reactions were conducted for 2 hours at 4 °C with continuous gentle rotation. NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs) was used for library construction.