NextSeq 500 sequencing; Ribosome profiling of astrovirus-infected cells
Ribosome profiling of astrovirus-infected cells
At 12 h post infection, cells were either not treated (NT) or treated with 50 µM LTM for 30 minutes, flash frozen in a dry ice/ethanol bath, and lysed in the presence of 100 µg/ml cycloheximide. Caco2 cells were grown on 150-mm dishes to reach 80-90% confluency, then infected at a multiplicity of infection (MOI) of 5 with HAstV1 virus stock (passage 2, derived from pAVIC1 T7 RNA; Geigenmüller et al., 1997, PMID 8995706).  After flash freezing, cells were transferred to dry ice and 400 µl of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide and 25 U/ml TURBO™ DNase (Life Technologies)] dripped on. Cells were scraped extensively to ensure lysis, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4 ºC, the supernatants recovered and stored in liquid nitrogen. Cell lysates were subjected to ribosome profiling based on previously described protocols (Chung et al., 2015, PMID 26286745; Irigoyen et al., 2016, PMID 26919232), except Ribo-Zero Gold rRNA removal kit (Illumina), not DSN, was used to deplete ribosomal RNA, and a 7-nt randomized sequence was appended to the 5' end of every read during library preparation.