RNA-Seq of macaca fascicularis: adult female ovarian microvascular endothelial cells
Total RNA was isolated from confluent mOMECs (n=4 animals) using the RNeasy Plus Mini Kit (Qiagen) using DNase (Qiagen) as per the manufacturers instruction. Total RNA quality was assessed using the Bioanalyzer RNA Nano chip kit (Agilent, Santa Clara, CA). RNA-seq and analysis were performed at the Roy J. Carver Biotechnology Center, University of Illinois. The RNAseq libraries were prepared with Illuminas TruSeq Stranded mRNAseq Sample Prep kit (Illumina, San Diego, CA). The libraries were pooled, quantitated by qPCR, and sequenced on two SP lanes for 151 cycles from both ends of the fragments on a NovaSeq 6000 (Illumina). Salmon  (v1.2.1) was used to quasi-map reads to the National Center for Biotechnology Informations Macaca fascicularis Annotation Release 101 transcriptome using macFas5 genome as the decoy sequence and options --seqBias --gcBias --numBootstraps=30 --validateMappings. Gene-level counts were then estimated based on transcript-level counts using the bias corrected counts without an offset method from the tximport package  in R (v4.1.0). The detection threshold was set at 0.5 cpm (counts per million) in at least four samples, which resulted in 13,715 genes being filtered out, leaving 14,451 genes to be analyzed for differential expression. After filtering, the trimmed mean of M (TMM) normalization was performed and normalized log2-based cpm values (logCPM) were calculated using edgeR. Differential gene expression analysis was performed using the limma-trend method using a model of Group + Batch. Batch was considered for sample batch effect. A global false discovery rate (FDR) correction was done across all contrasts at once so that the same raw P-value in any contrast ends up with the same FDR P-value.