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.本数据集针对从野生型（wild type, WT）或Nfkb2基因敲除（Nfkb2-/-）小鼠中分离的体外（Ex vivo）调节性T细胞（Treg cells）开展RNA测序（RNAseq）分析。实验设计：按照前述方法，从双亲代骨髓嵌合体小鼠的脾脏中分离野生型与Nfkb2-/- Treg细胞。分选得到的细胞于完全RPMI培养基中，在37℃条件下静置2小时，随后重悬于1ml TRIzol试剂（赛默飞世尔科技，加利福尼亚州）中并置于-80℃保存。本实验共设置3次生物学重复，分别收集野生型与Nfkb2-/- Treg细胞样本。按照TRIzol试剂推荐的RNA提取流程，使用氯仿、异丙醇与乙醇完成总RNA提取。提取得到的总RNA通过NanoDrop1000（赛默飞世尔科技，加利福尼亚州）进行浓度与纯度定量。按照试剂盒操作流程，使用TruSeq RNA样本制备试剂盒v2（Illumina，因美纳，圣迭戈）构建非链特异性RNA测序文库。将带有条码标签的测序文库在Illumina HiSeq 2500测序平台上完成测序。