title: Illumina HiSeq 2000 sequencing; Analysis of coronavirus and infected host-cell gene expression through RNA sequencing and ribosome profiling
design: Analysis of coronavirus and infected host-cell gene expression through RNA sequencing and ribosome profiling
library_construction_protocol: Murine 17 clone 1 (17Cl-1) (Sturman and Takemoto, 1972) and BHK-21 cells were maintained in Dulbeccos modification of Eagles 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 Hanks 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 CHX (Sigma-Aldrich; to 100 g/ml; 2 min), or HAR (LKT laboratories; 2 g/ml, 3 min) then CHX (to 100 g/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 l of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 g/ml cycloheximide and 25 U/ml TURBOTM 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 ribosomal RNA contamination was removed by treatment with duplex-specific nuclease (DSN) and library amplicons were constructed using a small RNA cloning strategy (Guo et al., 2010) adapted to Illumina smallRNA v2 to allow multiplexing. The methods used were as described by Chung et al. (2015), with minor modifications for the analysis of ribosomal pausing at the MHV 1 PRF signal (sample 40), namely a broader range of RPFs, migrating between 28 and 80 nt, were harvested prior to amplicon construction, and longer PCR amplicons of ~150-206 bp were gel purified.
attributes.tag: Experimental Factor: MHV-A59
attributes.value: infect
attributes.tag: Experimental Factor: cycloheximide
attributes.value: compound
attributes.tag: Experimental Factor: 5
attributes.value: time point