Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response

Sneha Rath; Eliza Prangley; Jesse Donovan; Kaitlin Demarest; Yigal Meir; Ned Wingreen; Alexei Korennykh

Gel bands were digested using 1.5 g Trypsin (Promega). Samples were dried completely in a SpeedVac and resuspended with 21 l of 0.1% formic acid (pH 3). Next, 5 l was injected per run using an Easy-nLC 1200 UPLC system. Samples were loaded directly onto a 45 cm long 75 m inner diameter nano capillary column packed with 1.9 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/484675 doi: bioRxiv preprint m C18-AQ (Dr. Maisch, Germany) mated to metal emitter in-line with an Orbitrap Fusion Lumos (Thermo Scientific, USA). The mass spectrometer was operated in data dependent mode with the 120,000 resolution MS1 scan (AGC 4e5, Max IT 50ms, 400-1500 m/z) in the Orbitrap followed by up to 20 MS/MS scans with CID fragmentation in the ion trap. Dynamic exclusion list was invoked to exclude previously sequenced peptides for 60s if sequenced within the last 30s and maximum cycle time of 3s was used. Peptides were isolated for fragmentation using the quadrupole (1.6 Da) window.