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

Following chromatin isolation, 50 µL of chromatin was mixed with 50 uL 2X run-on reaction mix ( The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 //doi.org/10. /2020 Inhibitor, 1% sarkosyl). The run-on reaction was performed at 37°C for 5 minutes at 700 rpm and stopped by adding 500 µL Trizol LS (Thermo Fisher Scientific, 10296-010) to the reaction. RNA samples were precipitated and resuspended in diethylpyrocarbonate (DEPC) treated water, heat treated at 65°C for 40 seconds, and digested on ice with 0.2N NaOH for 4 minutes. Base hydrolysis by NaOH was excluded from leChRO-seq protocols. Nascent RNA was purified with streptavidin beads (New England Biolabs (NEB), Ipswich, MA, S1421S) as previously described (Chu et al., 2018; Mahat et al., 2016) . RNA was purified from beads using Trizol (Thermo Fisher Scientific, 15596-026) and 3' adaptor ligation was performed with T4 RNA Ligase 1 (NEB, M0204S). Streptavidin bead binding was performed again following by 5' decapping with RNA 5' pyrophosphohydrolase (RppH, NEB M0356S). The 5' end of the RNA molecule was phosphorylated with T4 polynucleotide kinase (PNK, NEB M0201S) and 5' adaptor ligation was performed with T4 RNA Ligase 1. The 5' adaptor contained a 6-nucleotide unique molecular identifier (UMI) to allow for bioinformatic detection and elimination of PCR duplicates. Streptavidin bead binding was performed again followed by reverse transcription using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, 18090010). cDNA was amplified by PCR using the Q5 High-Fidelity DNA Polymerase (NEB, M0491S) to generate (le)ChRO-seq libraries. Libraries were sequenced (5' single end) at the Biotechnology Research Center at Cornell University on the NextSeq500 (Illumina, San Diego, CA). Primer sequences used for (le)ChRO-seq library preparation are provided in Table S8 .