title: NextSeq 500 paired end sequencing; Paired-end ribosome profiling of MARC-145 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV)-2, harvested at 9 hpi
design: Paired-end ribosome profiling of MARC-145 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV)-2, harvested at 9 hpi
library_construction_protocol: MARC-145 cells, a derivative of the MA-104 cell line, were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with L-glutamine (1 mM), antibiotics, and foetal bovine serum (FBS) (7%), at 37°C and 5% CO2. Cells were verified as mycoplasma-free by PCR (Universal Mycoplasma Detection Kit, ATCC) and deep sequencing. Cells were infected with NA PRRSV isolate SD95-21 (GenBank accession KC469618.1), or a mutant (KO2) based on this background. KO2 has been previously characterised (Fang et al., 2012; Li et al., 2014; Li et al., 2018), and has a modified nsp2 slippery sequence (G_GUU_UUU  to G_GUA_UUC), a premature stop codon 5 nt downstream in the –2 frame, and several mutations in and around the conserved C-rich motif (U3886A, U3889C, C3895A, C3898G, U3899A, C3900G, C3901U, C3905U and C3907G), where all mutations are synonymous in the ORF1a reading frame. Confluent 10 cm2 dishes of MARC-145 cells were infected with WT or KO2 virus at a MOI of 5, or mock-infected. At 9 hpi, cells were washed with 5 ml warm PBS and snap-frozen in liquid nitrogen. Snap-frozen dishes were transferred to dry ice and 400 µl 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)] added. The dish was transferred to ice to defrost, and then cells were scraped off the plate and triturated 10 times with a 26-G needle, before cell debris was removed by centrifugation (microfuge, 4°C, 20 min) and the supernatant was harvested and stored at −70°C. RNase I was added to the lysates for 45 minutes at room temperature, following which ribosomes and ribosome-protected fragments of RNA were purified by centrifugation through a sucrose cushion. RNA was extracted from the purified ribosomes by proteinase K digestion (enzyme from NEB, reaction at 42 degrees for 30 minutes) followed by hot acidic phenol:chloroform extraction and ethanol precipitation. 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 (Chung et al., 2015). Ribosomal RNA was removed using Ribo-Zero Gold rRNA removal kit (Illumina), and remaining RNA was purified by 15% denaturing urea gel purification to select fragments of 19-80nt. Library amplicons were constructed using adapters based on the TruSeq small RNA adapters, with an additional seven random nucleotides at the 5′-end of the 3′-adapter and the 3′-end of the 5′-adapter.