NextSeq 500 sequencing; RiboSeq of murine 17Cl-1 cells infected at MOI 10 with MHV-A59, compared to mock-infected controls, harvested at 5 and 8 hpi, plus a control tunicamycin-treated sample.
RiboSeq of murine 17Cl-1 cells infected at MOI 10 with MHV-A59, compared to mock-infected controls, harvested at 5 and 8 hpi, plus a control tunicamycin-treated sample.
8hpi_infected_s
17 Cl-1 cells were grown on 100-mm dishes to 90% confluency and infected with MHV-A59 at a multiplicity of infection (MOI) of 10, or treated with tunicamycin for 6 hours at 2ug/mL. At indicated h p.i., cells were rinsed with 5 ml of ice-cold PBS, flash frozen in a dry ice/ethanol bath and lysed with 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)]. The cells were scraped extensively to ensure lysis, collected and triturated ten times with a 26-G needle. Cell lysates were clarified by centrifugation at 13,000 g for 20 min at 4°C and then stored in liquid nitrogen. RNase I was added to the lysates for 45 minutes at room temperature, following which ribosomes and ribosome-protected fragments of RNA were purified by centrifugation through a sucrose cushion. RNA was extracted from the purified ribosomes by proteinase K digestion (enzyme from NEB, reaction at 42 degrees for 30 minutes) followed by hot acidic phenol:chloroform extraction and ethanol precipitation. Due to the very large amounts of vRNA produced during infection, mock samples were processed separately from infected samples, at all stages of nucleic acid extraction and library construction, to avoid contamination.  The methodologies employed were based on the original protocols of Ingolia and colleagues (Ingolia et al., 2009; Ingolia et al., 2012), except library amplicons were constructed using a small RNA cloning strategy (Guo et al., 2010) adapted to Illumina smallRNA v2 to allow multiplexing. Ribosomal RNA was removed using Ribo-Zero Gold rRNA removal kit (Illumina), and remaining RNA was purified by 15% denaturing urea gel purification to select fragments of 25-35nt, and library amplicons were constructed.