Hotspots of aberrant enhancer activity in fibrolamellar carcinoma reveal molecular mechanisms of oncogenesis and intrinsic drug resistance

Timothy A. Dinh; Ramja Sritharan; F. Donelson Smith; Adam B. Francisco; Rosanna K. Ma; Rodica P. Bunaciu; Matt Kanke; Charles G. Danko; Andrew P. Massa; John D. Scott; Praveen Sethupathy

ChRO-seq was performed as previously described (Chu et al., 2018; Mahat et al., 2016) with minor modifications. Length extension ChRO-seq (leChRO-seq) was performed identically to ChRO-seq except where indicated. Chromatin was isolated from pulverized frozen tissue in 1 mL 1X NUN buffer (20 mM HEPES, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1M urea, 1% NP-40, 1 mM DTT, 50 units/mL SUPERase In RNase Inhibitor (Thermo Fisher Scientific, Waltham, MA, AM2694), 1X Protease Inhibitor Cocktail (Roche, 11873580001)). For leChRO-seq, 50 units/mL RNase Cocktail Enzyme Mix (Thermo Fisher Scientific, AM2286) was substituted for SUPERase In RNase Inhibitor. Samples were vortexed for 1 minute, an additional 500 µL of 1x NUN buffer was added to each sample, and the samples were vortexed for an additional minute. Samples were incubated in an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany) at 12°C and shaking at 2000 rpm for 30 minutes before centrifugation at 12,500 x g for 30 minutes at 4°C. Each sample was washed with 1 mL 50 mM Tris-HCl (pH 7.5) supplemented with 40 units/mL SUPERase In RNase Inhibitor (80 units/mL for leChRO-seq) and centrifuged at 10,000 x g for 5 minutes at 4°C. This wash step was repeated twice and samples were stored in 50 µL of chromatin storage buffer (50 mM Tris-HCl pH 8.0, 25% glycerol, 5 mM magnesium acetate, 0.1 mM EDTA, 5 mM DTT, and 40 units/mL SUPERase In RNase Inhibitor). Samples were loaded into a Bioruptor (Diagenode, Denville, NJ) and sonicated on the high power setting for a cycle time of 10 minutes, consisting of 10 cycles of 30 seconds on and 30 seconds off. Sonication was repeated as necessary to solubilize the chromatin and samples were stored at -80°C.