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Sample GSM4809748

Query DataSets for GSM4809748

Status

Public on Sep 30, 2020

Title

A10_PRE

Sample type

SRA

Source name

PBMCs

Organism

Equus caballus

Characteristics

tissue: PBMCs
time point: PRE
horse id: A

Treatment protocol

PBMCs were prepared by Ficoll density gradient and lysed in Trizol. Liver biopsies were in RNAlater.

Extracted molecule

polyA RNA

Extraction protocol

Liver biopsies (~10 mg) fixed in RNA later were transferred into a MagNA Lyser Green Beads tube containing 1 mL cold TRIzol. Liver tissue was homogenized using a MagNA Lyser instrument (Roche) at 6500 rpm for 100 s with continuous cooling on ice every 20 s. PBMCs were directly lysed in TRIzol Reagent (Thermo Fisher Scientific). After addition of chloroform and centrifugation, the aqueous phase was mixed with 450 μL anhydrous ethanol and transferred to an RNA Clean & Concentrator-5 column (Zymo Research) for downstream RNA purification and concentration. The integrity of extracted RNA was analyzed on a 2100 bioanalyzer instrument using an RNA 6000 nano assay kit (Agilent Technologies).
The standard Illumina TrueSeq stranded mRNA library preparation protocol was followed for PBMC mRNA library preparation, starting from 500 ng input RNA. For the liver samples, total RNA-Seq was prioritized over poly(A) selection in order to recover viral reads. Depletion of ribosomal RNA from 500 ng input RNA was achieved using the Ribo-off rRNA depletion kit (Vazyme) for which horse rRNA removal has been validated. Following purification of rRNA-depleted total RNA using VAHTS RNA clean beads (Vazyme), the RNA was eluted into elution buffer ELB (Illumina) for subsequent TrueSeq stranded mRNA library preparation, starting from the mRNA fragmentation step. Quality of DNA libraries was checked using a 2100 bioanalyzer instrument. The Qubit dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific) was used to quantify DNA libraries in order to pool these in equimolar concentrations prior to denaturation. Pooled libraries were loaded on a NextSeq 500/550 v2.5 75 cycle flow-cell, and sequencing performed on a NextSeq benchtop sequencer (Illumina) at the Department of Clinical Microbiology (Hvidovre Hospital, Copenhagen). Data derived from two (PBMCs) or three (liver) independent deep-sequencing runs were pooled to ensure sufficient depth of sequencing coverage.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

For transcriptome analysis, reads were mapped to the EquCab3.0 genome (Ensembl v98) supplemented with the EqHV NZP1 genome (NC_038425) with HISAT2 and quantified with featureCounts including the positive and negative strand of annotation of NZP using default settings. Previous equine transcriptome analysis identified duplicate entries for numerous immunologically relevant genes (CD14, IL7R, etc.) in the EquCab3.0 reference. To recover quantification of genes with duplicate entries (n = 77 genes, which were discarded in initial quantifications due to read multimapping), featureCounts was run a second time on a filtered reference containing only genes with duplicate entries; in this stage, multimapped reads were counted fractionally (each alignment carries 1/x counts where x is the total number of alignments reported for the read) and then collapsed at the gene level by summation (featureCounts with -m and –fraction explicitly). The resulting counts matrix for genes with duplicate entries was appended to the initial count matrix. Differential expression was analyzed in R using Limma and Voom. The PRE, ACUTE, SC, FV and POST time points were included in analysis. Horse D was excluded due to persistent infection. Horse B was excluded from liver but not PBMC analysis for technical reasons.
Genome_build: EquCab3.0
Supplementary_files_format_and_content: PBMC Counts matrix. RNA-seq raw read counts for all individual samples are given across genes. Annotations are by Ensembl Gene ID, except for genes found to be multi annotated in which case annotations are by gene names.
Supplementary_files_format_and_content: Liver Counts matrix. RNA-seq raw read counts for all individual samples are given across genes. Annotations are by Ensembl Gene ID, except for genes found to be multi annotated in which case annotations are by gene names.
Supplementary_files_format_and_content: PBMC Voom limma AllTableF. Normalized abundance measurements as determined by voom/limma modelling. For comparison across all time points (F-test), the following is shown: ensembl_gene_id, ensembl_transcript_id, external_gene_name, entrezgene_id, chromosome_name, strand, the normalized expression values for the time points Acute, Seroconversion (SC), Clearance/Falling Viremia (FV), and Post, the average normalized expression values (AveExpr), F, P.Value and adj.P.Val (FDR).
Supplementary_files_format_and_content: Liver Voom limma AllTableF. Normalized abundance measurements as determined by voom/limma modelling. For comparison across all time points (F-test), the following is shown: ensembl_gene_id, ensembl_transcript_id, external_gene_name, entrezgene_id, chromosome_name, strand, the normalized expression values for the time points Acute, Seroconversion (SC), Clearance/Falling Viremia (FV), and Post, the average normalized expression values (AveExpr), F, P.Value and adj.P.Val (FDR).

Submission date

Sep 29, 2020

Last update date

Oct 01, 2020

Contact name

Troels K H Scheel

E-mail(s)

tscheel@sund.ku.dk

Organization name

University of Copenhagen

Department

Institute for Immunology and Microbiology