RNA-Seq Analysis of Sorted Cell Populations

Total RNA was extracted from flow cytometry–sorted cells using QIAshredders and an RNeasy Plus Micro kit (QIAGEN) (25 (link), 45 (link)). RNA quality and quantity were analyzed using a NanoDrop and Bioanalyzer. RNA-Seq library preparation and sequencing were conducted at the Genomics and Microarray Core at the University of Colorado Anschutz Medical Campus (46 (link)). Libraries were constructed using a SMARTer Stranded Total RNA-Seq kit (Clontech) customized with mouse-specific oligonucleotides for rRNA removal. Directional mRNA-Seq of sorted cell populations was conducted using the Illumina HiSeq 2500 or 4000 system.