Sample 7
Mus musculus
Protocols: Murine 17 clone 1 (17Cl-1) (Sturman and Takemoto, 1972) cells were maintained in Dulbecco's modification of Eagle's medium supplemented with 10% (vol/vol) fetal calf serum (FCS). Recombinant MHV strain A59 (MHV-A59) was derived as previously described (Coley et al., 2005). 17Cl-1 cells (10^7) were plated in 10 cm dishes and, upon reaching 70-80% confluence, were infected with MHV-A59 at a multiplicity of infection (MOI) of 10 PFU/cell in Hank's balanced salt solution (HBSS) containing 50 mg/ml DEAE-dextran and 0.2% bovine serum albumin (BSA). After 45 min at 37 ºC, the inoculum was removed and the cells were incubated in DMEM containing 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 ºC until harvest. At the appropriate time point, cells were flash frozen or treated with 1 X CHX (to 100 µg/ml; 2 min), or 100 X CHX (to 10,000 µg/ml; 2 min). Cell lysates were subjected to RiboSeq and RNASeq. 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.