PAR-iCLIP MCMV infection

Swiss murine embryonic fibroblasts (NIH-3T3) or murine TCMK-1 epithelial cells were infected with BAC-derived wild-type Smith strain mouse cytomegalovirus (MCMV) at a multiplicity of infection (MOI) of 10. PAR-iCLIP was performed using 4-thiouridine labeling on both uninfected and MCMV-infected cells (72h post infection). NIH-3T3 or TCMK-1 murine epithelial cells from seven 15 cm dishes per condition (for MCMV infection) were trypsinized and resuspended in 1ml cell culture medium per plate. Cells were infected immediately after trypsinization in suspension (7ml) with wild-type MCMV at an MOI of 10. 1 h later, fresh cell culture medium was added (containing 25 µM 4-thiouridine (4sU)) and cells were plated on to ten 15cm dishes. After 24 and 48 h of infection, +25 µM and +50µM 4sU was added to the cell culture medium. For the uninfected samples, cells were plated onto fresh plates. 24h after platting, metabolic RNA labeling was performed for 24h using 100 µM of 4sU. Prior to UV cross-linking at 365 nm on ice, cell culture medium was taken off the plates. Cross-linking was performed at 0.15 J/cm2 of 365 nm UV light using a Spectrolinker XL-1500 (Spectronics Corporation). Cells were harvested and iCLIP libraries were prepared as described in detail (Konig et al., J Vis Exp. 2011). TCMK-1 murine kidney epithelial cells (ATCC: CCL-139) and M2-10B4 bone marrow stroma cells (ATCC: CRL-1972) were cultured in DMEM containing 10% fetal calf serum and penicillin/streptomycin. NIH-3T3 fibroblasts (ATCC: CRL-1658) were cultured in DMEM medium containing 5% fetal calf serum and penicillin/streptomycin. For MCMV infection, cells from seven 15 cm dishes (80% confluent) per condition (for MCMV infection) were trypsinized 3-4 days after the last split. Cells were resuspended in 1 ml cell culture medium per plate. MCMV virus stock was added at an MOI of 10 (t=0). Similar to centrifugal enhancement, infecting cells in suspension rather than on plates results improves infection by about 10-fold.). 1h later, cells were transfered to a bottle with 195 ml cell culture media containing a final concentration of 25 µM 4sU. Cells were plated to ten 15 cm dishes (20 ml per plate). At 24 and 48 h post infection, an additional 25µM (24hpi) and 50 µM (48hpi) 4sU was added to the cell culture medium. Uninfected cells were split 24h prior to beginn of 4sU labeling (100µM 24h).