GSE121685
Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17ß.
The goals of this study is to enrich the population of ovine gonadotropes via adenovirus-mediated targeting and flow cytometry to compare NGS-derived transcriptome profiling (RNA-seq) of a gonadotrope-enriched cell population to the combined transcriptome of all anterior pituitary cells  and apply enrichment method to query the transcriptome of sorted ovine pituitary cells in response to treatment with 10 nM estradiol (E2) for six hours. Methods: mRNA profiles of sheep anterior pituitary cells were generated by deep sequencing, in sextuplicates, using Illumina HiSeq. The sequence reads that passed quality filters were analyzed at the transcript level with GSNAP, followed by featureCounts and then followed by edgeR. qRT–PCR validation was performed using SYBR Green assays. Results: About 124 million sequence reads per sample were mapped to the sheep genome (Oar v.3.1) which identified 17,082 transcripts in the pituitaries. RNA-seq data confirmed overrepresentation of genes known to be expressed in pituitary in sorted samples; 11 of genes (5 gonadotrope-specific and 6 non gonadotrope-specific) were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR. Approximately 7.2% of the transcripts showed differential expression between the sorted (gonadotropes) and unsorted (all anterior pituitary cells) pituitary cells, with a fold change =3 and FDR <0.05. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to gonadotrope function. 232 genes were detected as upregulated in the sorted GFP-positive cells treated with E2 (gonadotropes) and 31 were downregulated following E2 treatment with a fold change =1.5 and FDR <0.1. Conclusions: Our study represents the first detailed analysis of the gonadotrope transcriptome, with 6 biologic replicates, generated by RNA-seq technology. The data analysis workflows reported here should provide a framework for comparative investigations of gonadotrope expression profiles. This method was applied to determine differentially expressed genes in the sorted population of pituitary cells (gonadotropes) in response to 10 nM E2. Overall design: Pituitary mRNA profiles from primary ovine cultures infected with virus specifically labeling gonadotropes (cont-sorted), sorted cells treated with E2 (e2-sorted), unsorted (anterior pituitary cells) (cont-unsorted) and unsorted treated with E2 (e2-unsorted) were generated by deep sequencing, in sextuplicates, using Illumina HiSeq.