Dataset Information

High throughput sequencing of MARC145, A549, HEK293, and HRT18 cells infected with Shaan virus

ABSTRACT: Purpose: The goals of this study are to monitor the evolution pattern of Shaan virus in depending host cells by viral transcriptome sequencing analysis of MARC145, A549, HEK293, and HRT18 cells infected with Shaan virus. Methods: The original isolate of Shaan virus (B16-40, Genbank accession no. MG230624.1) was passaged in MARC-145 cells. Low-passaged virus was obtained from the virus 4 times serially passaged in MARC-145 cells. The shaan virus which was 44 times serially passaged in MARC-145 cells was used for high-passaged virus. The prepared low- (p4) and high passaged shaan virus (p44) in MARC-145 cells were inoculated in triplicate to different host cells, HEK-293, A549, and HRT18 cell lines at M.O.I of 0.5 for 2 hours. The infected cells were incubated with the maintenance medium for each cell line for 24hrs. The infected cell pellets were resuspended to 250µl with fresh medium, to extract RNA for the high-throughput sequencing. The RNA samples were sequenced with illumine TruSeq Strand Total RNA LT kit and illumine NovaSeq6000 plaform form Macrogen, Inc (Seoul, Korea) for high throughput sequencing. The raw reads were trimmed with BBDuk and mapped the isolate Bat-ParaV/B16-40 (Genebank accession number. MG230624.1) with Bowtie 2 using Geneious program 2021.2.2. Transcript expression level in cells infected with shaan virus were calculated based on annotation on a reference shaan virus. Result: The total reads counts were between 50,285,454 and 76,298,278. The reference mapping of the trimmed reads with a reference genome (Genbank accession no. MG230624.1) showed different coverage values. The mapped reads were normalized and expressed as Transcript per Million (TPM) value. There were no noticeable differences of TPM values of each gene between the cells infected with low- and high-passaged shaan virus. However, the nucleocapsid (N) and matrix (M) gene-associated transcripts were shown to be differently measured among the host cells. Conclusion: In this regard, we tried to investigate certain selected mutation patterns by host switching using shaan virus isolate in MARC-145 cells. Therefore, this study provided potential evidence for host-specific selective mutation patterns by cell types as well as host of cells for shaan virus evolution.

ORGANISM(S): Chlorocebus aethiops Homo sapiens

PROVIDER: GSE201344 | GEO | 2022/04/30

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Purpose: The goals of this study are to monitor the evolution pattern of SARS-CoV2 in depending host cells by viral transcriptome sequencing analysis of Vero, A549, Caco2, and HRT18 cells infected with SARS-CoV2. Methods: SARS-CoV-2 isolate was passaged 4 time on Vero cells and used to extract RNA for the high-throughput sequencing. The 8×104 PFU of SARS-CoV2 stocks passaged on vero cells were inoculated to the monolayer of A549, CaCO2, and HRT-18 cell lines in 75T flask for 1hour at 37℃ in a 5% CO2 incubator with gentle shaking of 15 minutes interval. After that, the infected cells were washed two times with DPBS and incubated with the fresh maintenance medium for 3 days. The virus inoculation was performed in triplicate for each cell lines. In case of the first passage, the infected cell pellets were resuspended to 250µl with fresh medium, to extract RNA for the high-throughput sequencing. The cultured cell supernatant of the virus-infected A549, CaCO2, and HRT18 cells was centrifuged at 3,000g for 10min to use for the next passage, and stored at -80℃. The serial passage of SARS-CoV-2 on A549, CaCO2, and HRT18 cell lines were continued to passage 12 and the cultured cell supernatant of the infected cells in passage 12 was centrifuged at 3,000g for 10 min, and used to extract RNA for the high-throughput sequencing. The RNA samples were sequenced with illumine TruSeq Strand Total RNA LT kit and illumine NovaSeq6000 plaform form Macrogen, Inc (Seoul, Korea) for high throughput sequencing. The raw reads were trimmed with BBDuk and mapped the isolate SARS-CoV-2/human/KOR/KCDC03-NCCP43326/2020 (Genebank accession number. MW466791) with Bowtie 2 using Geneious program 2021.2.2 Result: Using SNP analysis workflow, our result showed the sequence variations pattern of SARS-CoV2 depending on host cell (A549, CaCO2, and HRT18 cell lines) and it was confirmed that a relatively large number of SNPs were commonly observed in spike protein. Some SNPs affect amino acid changes, and a common pattern of amino acid changes was observed the genomic sequence of SARS-CoV2 passaged in A549, CaCO2 and HRT18 cells. Conclusion: In this study, we tried to monitor the SARS-CoV-2 (GenBank accession No. MW466791 in 2020, Korea) evolution pattern in different host cells using high throughput sequencing analysis, and compare the selected mutations by each host cells with natural mutations found in currently circulating SARS-CoV-2 variants.

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

High throughput sequencing of MARC145, A549, HEK293, and HRT18 cells infected with Shaan virus

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets