title: NextSeq 500 sequencing; Ribosome profiling of Japanese encephalitis virus infected Neuro2a cells
library_construction_protocol: Cells were rinsed with ice-cold PBS containing 100ug/ml cycloheximide. Dishes were submerged in a liquid nitrogen reservoir for 10s followed by scraping over dry ice in polysome lysis buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 5mM MgCl2, 1mM DTT, 1% (v/v) Triton X-100, 100ug/ml cycloheximide and 25U/ml TURBO DNase I (Life Technologies)). Neuro2a cells were maintained in Dulbecco's modification of Eagle's medium (DMEM, Himedia) supplemented with 10% (v/v) foetal bovine serum (FBS, Gibco) and 1% (v/v) Penicillin-Streptomycin (Sigma). Cells were grown to 70% confluence in 10cm dishes and infected with 5 plaque forming units (PFU) per cell of JEV P20778 Vellore strain (AF080251) or Dimethyl sulfoxide (DMSO, mock) in plain DMEM. After 60 min at 37 degrees, inoculum was removed, and monolayer was extensively washed with phosphate buffered saline (PBS) before replacing with complete media (DMEM with 10% FBS). At 18 hours post infection (hpi), cells were treated with either cycloheximide (Sigma-Aldrich, 100ug/ml, 5min) or harringtonine (LKT laboratories, 2ug/ml, 2min) followed by cycloheximide treatment for 5min. Cells were collected and triturated with a 26G needle 20 times and clarified by centrifugation (13000g, 20min, 4degrees). Supernatant was collected and treated with 100U/ul of RNase I (Ambion) for 1h at room temperature with gentle mixing followed by inactivation with 40U of SUPERase-In RNase inhibitor (Ambion). Cell extracts were passed through Sephacryl S400 spin columns (GE) after pre-equilibration with polysome lysis buffer and ribosome-bound mRNA eluates were collected by centrifugation at 600g, 1min, 4degrees. For RNA-Seq, total RNA was fragmented using 10x fragmentation reagent (Ambion) according to manufacturer's protocol. RNA was extracted from eluates and total lysate using TRIzol reagent (ThermoScientific). Purified RNA was size selected for a broader range of fragments (25 to 70 nucleotides) and subjected to RiboSeq and RNASeq library preparation based on original protocols (Ingolia et al., 2012). Final amplicons were PCR amplified for 6, 8 and 10 cycles and pooled before sequencing.