NextSeq 500 sequencing; RNASeq of murine 17Cl-1 cells infected at MOI 10 with MHV-A59, compared to mock-infected controls, harvested at 5 and 8 hpi.
RNASeq of murine 17Cl-1 cells infected at MOI 10 with MHV-A59, compared to mock-infected controls, harvested at 5 and 8 hpi.
5hpi_mock_2_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. 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, the supernatants recovered and stored in liquid nitrogen. RNA was extracted from the lysates 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 fragmented by alkaline hydrolysis (45mM NaHCO3, 6mM Na2CO3, 1mM EDTA; 95 degrees, 15 minutes). Fragments of 25-35nt were selected by 15% denaturing urea gel purification, and library amplicons were constructed.