GSM3178095: RBMX_SEC_fraction20; Homo sapiens; RIP-Seq
To prepare cell lysates, pellets were thawed on ice and resuspended in NP40 lysis buffer (50 mM HEPES pH 7.5, 150 mM KCl, 2 mM EDTA, 1% (v/v) NP40, 0.25 mM DTT, complete EDTA-free protease inhibitor cocktail) and incubated on ice for 10 min. We sonicated cell lysates using a Branson Digital Sonifier with a microtip set at 5 W power for a total of 1 min 30 s in intermittent pulses (0.7 s on, 2.3 s off), followed by RNase I (Thermo Fisher Scientific) digestion (0.5 U/µl, 10 min at 23 °C). Subsequently, we added 15 µl/ml Murine RNase Inhibitor (New England Biolabs), followed by DNA digestion (20 U TURBO DNase (2 U/µl; Thermo Fisher Scientific), 2.5 mM MgCl2 and 0.5 mM CaCl) for 20 min at 37 °C. We incubated samples on ice for 10 min before clearing lysates by centrifugation at 15,000g for 15 min. Insoluble material was removed and total protein concentration was determined by BCA assay. Cell lysates were flash-frozen and stored in batches of 10 mg total protein at -80 °C. For each immunoprecipitation experiment, lysates (10 mg total protein) were thawed on ice and pre-cleared by incubating with Protein A/G magnetic beads (using 30 µl/mg total protein) for 30 min at 4 °C. Immunoprecipitation reactions were performed as described in Hafner et al. 2010 Cell 141, 129–141. Sequencing libraries were constructed as described in Zarnegar et al. 2016 Nat Meth 13, 489–492, with the exception of reverse transcription reactions, which were carried out as described in Hafner et al. 2010 Cell 141, 129–141. Size-matched input libraries were constructed as described in Van Nostrand et al. 2016 Nat Meth 13, 508–514.