NextSeq 500 paired end sequencing; RNA sequencing of primary Naive CD4+ T cells and Activated Naive CD4+ T cells
naive CD4+ T cells have been isolated from peripheral PBMCs by density gradient centrifugation with Ficoll-Paque Plus. From PBMCs, T cells were negatively selected with magnetic separator (AutoMACS). Naive CD4+ were FACS sorted as CD4+CD25-CD127-/highCD45RO-. Activation to Th1 phenotype with IL-2, IL12 and anti-IL4 and stimulated with Dynabeads Human T-activator anti-CD3/anti-CD28 beads for 16 hours Total, Chromatin and Nucleoplasm RNAs have been extracted with Maxwell RSC miRNA Tissue kit  RNA was ribodepleted with RiboGone-Mammalian and the library preparation was executed with SMARTer Stranded RNA-Seq kit