Illumina HiSeq 2000 paired end sequencing; GSM1829780: CON_THY_Rep1; Sus scrofa; RNA-Seq

CON_THY_Rep1

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of 3rd trimester pregnant pigs can result in transmission of the virus to the fetus and ultimately death in utero or postnatally. Little is known about the immune response to infection at the maternal-fetal interface and in the fetus itself, or the molecular events behind virus transmission and disease progression in the fetus. To investigate these processes, RNA-sequencing of two tissues, uterine endothelium adjacent to the umbilical attachment site and fetal thymus, was performed 21 days post challenge on four groups of fetuses selected from a large PRRSV challenge experiment of pregnant gilts. Overall design: RNA-seq experiment compared gene expression between four different groups of fetuses (n=12 per group): control (CON-uninfected fetuses from mock inoculated gilts), UNINF (uninfected fetuses from PRRSV-inoculated gilts), INF (infected fetuses from PRRSV-inoculated gilts), and meconium-stained fetuses (MEC-meconium-stained fetuses from PRRSV-inoculated gilts) and investigated two tissues: uterine endometrium (with adherent placental tissue) at the site of umbilical attachment and fetal thymus (96 samples in total). Three contrasts were performed for the differential expression (edgeR) and network (WGCNA) analyses: UNINF v CON, INF v UNINF, and MEC v INF.

Genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus

Submitted by Gene Expression Omnibus on 18-MAY-2016

Total RNA was isolated from 10-20 mg of endometrium and thymus of each fetus using an ‘All Prep RNA/DNA isolation Mini kit’ (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was quantified by spectrophotometry using a Nanodrop ND 2000 (Thermo Fisher Scientific, Waltham, USA). RNA quality was assessed by digital electrophoresis using a ‘2200 Tapestation’ (Agilent Technologies, Santa Clara, USA). The mean RNA Integrity Number (RIN) sample value was 9.0 for thymus (range 8.0-9.8) and 8.3 for endometrium (range 7.5-8.8). A total of 96 cDNA libraries were constructed for RNA-sequencing (RNA-seq) from thymus and endometrium samples of individual fetuses. Libraries were constructed from 1 μg of input total RNA using the TruSeq RNA sample preparation kit v2 (Illumina, San Diego, USA) according to the manufacturer’s instructions. DNA libraries were sequenced at the Genome Quebec Innovation Centre (McGill University, Montreal, Canada). Library insert size distribution was assessed on a LabChip GX instrument (Perkin Elmer, Waltham, USA) using a high sensitivity chip. Library concentration was determined using a KAPA Library Quantification Kit (KAPA Biosystems) on a Lightcycler 480 II instrument (Roche Life Science, Indianapolis, USA). Cluster formation was performed on the cBot instrument (Illumina) with eight libraries per lane (7 pM per library) using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Paired-end 100 bp cycle sequencing was performed on a HiSeq 2000 (Illumina) using the TruSeq SBS kit v3-HS (Illumina).

Genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus.

Genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus

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