title: NextSeq 500 sequencing; Ribosome profiling of MARC-145 or MA-104 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV)-1 or PRRSV-2, harvested at 3-12 hpi
design: Ribosome profiling of MARC-145 or MA-104 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV)-1 or PRRSV-2, harvested at 3-12 hpi
library_construction_protocol: For PRRSV-1-infected cells: MA-104 cells, a cell line derived from foetal kidney tissue of the African green monkey species Chlorocebus sabaeus, were infected with a PRRSV strain derived from the Porcilis® vaccine strain (MSD Animal Health; GenBank accession KJ127878.1) but with 148 accumulated mutations (see associated publication for genome sequence). Confluent 6 cm2 dishes of MA-104 cells were infected at a multiplicity of infection (MOI) within the range 1-3. At 0 (mock) and 8 hpi cycloheximide (Sigma-Aldrich) was added to the medium (final concentration 100 µg/ml) and incubated for 2 min. Cells were rinsed with 5 ml of ice cold PBS, placed on ice, and 400 µl lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml CHX and 25 U/ml TURBO™ DNase [Life Technologies] was added drop-wise. 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. For PRRSV-2-infected cells: 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. For the CHX_Ribo_9hpi libraries, CHX pre-treatment and harvesting was performed at 9 hpi as described for PRRSV-1-infected cells. For all other libraries, at 3, 6, 9, or 12 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 (composition as described above) added. The dish was transferred to ice to defrost, and then cells were scraped and processed as described above. 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 25-34 nt (PRRSV-1-infected samples, CHX-pre-treated PRRSV-2- or mock-infected samples, and non-CHX-pre-treated PRRSV-2- or mock-infected 9 hpi replicate one samples) or 19-34 nt (all other samples), from which library amplicons were constructed using adapters based on the TruSeq small RNA adapters. For all PRRSV-2-infected (or mock-infected) libraries, adapters with an additional seven random nucleotides at the 5′-end of the 3′-adapter and the 3′-end of the 5′-adapter were used to generate amplicons. For PRRSV-1 replicate one, no random nucleotides were present on the adapters, and for PRRSV-1 replicate two, 14 random nucleotides were present at the 5′-end of the 3′-adapter.