Characterization of DHX15 Interactome

HEK293T cells were transfected with a pool of siRNAs targeting DHX15 (Supplemental Table 19) using Lipofectamine RNAiMax (ThermoFisher Scientific) according to manufacturer instructions. Cells were transfected again after 24 hours and harvested at day 4. For overexpression experiments, HEK293T cells were transfected with pCMV6-DHX15 or pCMV6-DHX15 R222G. RNA was extracted after 48 hours with Trizol and libraries prepared as described above. Pull down experiments for mass spectrometry were done using the Pierce c-Myc Tag IP/Co-IP Kit (Thermo Scientific). The MS analysis was performed according to the optimized platform as previously reported^{74 (link)}. Proteins in the gel band were in-gel digested by trypsin. Resulting peptides were loaded on a nanoscale reverse-phase column, and eluted by a gradient (~30 min). Eluted peptides were detected by an inline LTQ Orbitrap Elite mass spectrometer (Thermo Scientific). Acquired data were searched against the protein database using the Sequest algorithm and filtered to reduce the protein false discovery rate to below 1%. For binary comparison, statistical analysis was also performed based on the G-test. FDR was derived according to biological replicates and null hypothesis^{75 (link)}.