ERS6987124 SAMEA9252521 60711 Chlorocebus sabaeus Protocols: 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. ENA first public 2022-03-09 ENA last update 2022-03-09 External Id SAMEA9252521 INSDC center alias Division of Virology, Department of Pathology, University of Cambridge INSDC center name Division of Virology, Department of Pathology, University of Cambridge INSDC first public 2022-03-09T12:27:51Z INSDC last update 2022-03-09T12:27:51Z INSDC status public Submitter Id E-MTAB-10622:noCHX_Ribo_9hpi_KO2_2 broker name ArrayExpress cell line MARC-145 cell type kidney cell developmental stage late embryo disease normal infect PRRSV-2 KO2 organism part kidney sample name E-MTAB-10622:noCHX_Ribo_9hpi_KO2_2 ERS6987125 SAMEA9252522 60711 Chlorocebus sabaeus Protocols: 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. ENA first public 2022-03-09 ENA last update 2022-03-09 External Id SAMEA9252522 INSDC center alias Division of Virology, Department of Pathology, University of Cambridge INSDC center name Division of Virology, Department of Pathology, University of Cambridge INSDC first public 2022-03-09T12:27:51Z INSDC last update 2022-03-09T12:27:51Z INSDC status public Submitter Id E-MTAB-10622:noCHX_Ribo_9hpi_mock_2 broker name ArrayExpress cell line MARC-145 cell type kidney cell developmental stage late embryo disease normal infect mock organism part kidney sample name E-MTAB-10622:noCHX_Ribo_9hpi_mock_2 ERS6987126 SAMEA9252523 60711 Chlorocebus sabaeus Protocols: 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. ENA first public 2022-03-09 ENA last update 2022-03-09 External Id SAMEA9252523 INSDC center alias Division of Virology, Department of Pathology, University of Cambridge INSDC center name Division of Virology, Department of Pathology, University of Cambridge INSDC first public 2022-03-09T12:27:51Z INSDC last update 2022-03-09T12:27:51Z INSDC status public Submitter Id E-MTAB-10622:noCHX_Ribo_9hpi_WT_2 broker name ArrayExpress cell line MARC-145 cell type kidney cell developmental stage late embryo disease normal infect PRRSV-2 WT organism part kidney sample name E-MTAB-10622:noCHX_Ribo_9hpi_WT_2