Unknown,Transcriptomics,Genomics,Proteomics

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Ribosome profiling and RNA sequencing of ED fibroblast cells in response to infection by equine torovirus, Berne strain.

ABSTRACT: Ribosome profiling (Ribo-Seq) (maps positions of translating ribosomes on the transcriptome) and RNA-Seq (quantifies the transcriptome) analysis of equine torovirus.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Equus caballus

SUBMITTER: Andrew Firth

PROVIDER: E-MTAB-6656 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cell lines derived from Chlorocebus sabaeus kidney were infected with an isolate of PRRSV-1 or PRRSV-2 and ribosome profiling was performed (this entry) in parallel with RNASeq (see related accession number). These datasets were used to analyse the viral and host translatome, frameshifting on the viral genome, and putative frameshift-related ribosome pausing events. For the PRRSV-1 experiments, MA-104 cells were infected with an isolate based on the Porcilis vaccine strain (KJ127878.1 but with several accumulated mutations, see associated publication) and harvested at 8 hpi after pre-treatment with cycloheximide (CHX). For the PRRSV-2 experiments, MARC-145 cells were infected with SD95-21 PRRSV (KC469618.1), and a mutant thereof (KO2). One group of samples was harvested at 9 hpi after pre-treatment with CHX, and another group of samples was harvested at 3, 6, 9 and 12 hpi by flash-freezing without CHX pre-treatment. For all samples, RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 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 long (all other samples). Fragments were cloned into 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. 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. Libraries were sequenced on the Illumina NextSeq 500 platform as a single-end run. Non-CHX-pre-treated PRRSV-2-infected 9 hpi replicate two libraries were uploaded under a separate accession number due to differences in the size selection and sequencing protocol - see associated paired-end entry. Note that sample nomeclature (including replicate numbers) is consistent between this and the two related accessions, and RiboSeq libraries are matched with RNASeq libraries, which were prepared from the same lysate. The noCHX_Ribo_9hpi_mock_3 library is deliberately absent as this was a poor quality library.

Project description:Cell lines derived from Chlorocebus sabaeus kidney were infected with an isolate of PRRSV-1 or PRRSV-2 and RNASeq was performed (this entry) in parallel with ribosome profiling (see related accession numbers). These RNASeq datasets provide information on the transcriptome of PRRSV-infected cells and were used to detect novel viral transcripts and perform differential gene expression analysis on host transcripts. For the PRRSV-1 experiments, MA-104 cells were infected with an isolate based on the Porcilis vaccine strain (KJ127878.1 but with several accumulated mutations, see associated publication) and harvested at 8 hpi after pre-treatment with cycloheximide (CHX). For the PRRSV-2 experiments, MARC-145 cells were infected with SD95-21 PRRSV (KC469618.1), and a mutant thereof (KO2). One group of samples was harvested at 9 hpi after pre-treatment with CHX, and another group of samples was harvested at 3, 6, 9 and 12 hpi by flash-freezing without CHX pre-treatment. For all samples, RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 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 ~50 nt long (all other samples). Fragments were cloned into 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. 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. Libraries were sequenced on the Illumina NextSeq 500 platform as a single-end run. For one NextSeq run, some potyvirus amplicons, indexed using TruSeq small RNA indices 1-48, was spiked in to the pool directly before sequencing, therefore some libraries (noCHX_RNA_9hpi_KO2_1, noCHX_RNA_9hpi_KO2_2, noCHX_RNA_9hpi_mock_1, noCHX_RNA_9hpi_mock_2, noCHX_RNA_9hpi_WT_1, noCHX_RNA_9hpi_WT_2) have a small proportion of potyvirus reads, but this does not represent co-infection in the biological sample and does not affect the conclusions of the study. Note that sample nomeclature (including replicate numbers) is consistent between this and the two related accessions, and RiboSeq libraries are matched with RNASeq libraries, which were prepared from the same lysate.

Dataset Information

Ribosome profiling and RNA sequencing of ED fibroblast cells in response to infection by equine torovirus, Berne strain.

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