Name: Index1_s
Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Murine 17 clone 1 (17Cl-1) (Sturman and Takemoto, 1972) and BHK-21 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 (or 200 PFU/cell in the “High MOI” experiment - samples 37-39) 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 treated with Tunicamycin then 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). Cells were rinsed with 5 ml of ice-cold PBS, the dishes submerged in a reservoir of liquid nitrogen for 10 s, 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 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.