Role of NF-kappaB2-p100 in regulatory T cell homeostasis and activation

RNAseq analysis of Ex vivo Treg cells were isolated from wild type or Nfkb2-/- mice. Overall design: WT and Nfkb2-/- Tregs were isolated as described above from spleens of bi-parental bone marrow chimeras. Sorted cells were rested in complete RPMI at 37°C for 2 h before being re-suspended in 1ml TRIzol reagent (Thermofisher Scientific, CA) and stored at -80°C. Three such biological repeats of WT and Nfkb2-/- Tregs were collected. RNA was isolated by treatment with chloroform, isoporopanol and ethanol according to the RNA isolation protocol using TRIzol reagent as recommended. The RNA extracted was quantified using NanoDrop1000 (Thermofisher Scientific, CA). Unstranded RNAseq libraries were prepared using TruSeq RNA Sample Prep Kit v2 (Ilumina, San Diego) according to the kit protocol. Barcoded libraries were sequenced in Illumina HiSeq 2500.