RNA sequencing

RNA-sequencing (RNA-seq) analysis was performed by the Informatics Resource Center, Institute for Genome Sciences, University of Maryland School of Medicine. Paired end Illumina libraries were mapped to the mouse reference, Ensembl GRCm38.74, using TopHat v1.4.0 with the default mismatch parameters. Read counts for annotated genes were calculated using HTSeq. DESeq Bioconductor package v1.5.24 was used to normalize read counts by library size to generate gene counts per million, estimate dispersion, and determine differentially expressed genes between two groups. Downstream analysis was done with differentially expressed transcripts with a false discovery rate of ≤0.05 and log₂ fold change. Ingenuity Pathway Analysis (IPA) was used to compute enrichment of biological pathways using the list of differentially expressed genes. IPA was also used to analyze upstream regulators by predicting the regulators of each data set and their activation state. Each predicted upstream regulator is assigned a z-score for their activation state based on previous experimentally observed transcriptional events from the literature. A z-score of <2 predicts a regulator to be inhibitory, whereas a z-score of >2 indicates the regulator is activating. Additionally, each upstream regulator is assigned an overlap p value, a measure of statistically significant overlap between genes in the dataset and genes known to be regulated by this regulator. Reads were deposited in the National Center for Biotechnology Information Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA493118) under BioProject PRJNA493118,.