Naïve Regulatory T Cell Subset Is Altered in X-Linked Agammaglobulinemia (part2)

Clonosets from 3 cell subsets (CD4+ mature naive, CD4+ RTE, naive Treg) sorted from blood of XLA and healthy (old and young) donors. Total RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. In all experiments described here, cDNA libraries were obtained using 5’-RACE with unique molecular identifiers (UMI) using Human TCR profiling kit (MiLaboratory LLC). Libraries were sequenced on Illumina NextSeq 500 using 300 cycle reagent kit, paired-end 150 + 150 nt mode. Clonotypes were inferred from reads using MIGEC (UMI threshold = 2) + MIXCR + VDJTOOLS<br>

本数据集的克隆型集合（clonosets）源自3种细胞亚群：CD4+成熟初始T细胞、CD4+近期胸腺迁出细胞（RTE）、初始调节性T细胞（naive Treg），上述细胞均从XLA患者以及健康青年、老年供者的外周血中分选获得。总RNA提取严格遵循制造商操作说明，使用Qiagen公司的RNeasy迷你试剂盒完成。本研究所有实验均采用人类T细胞受体分型试剂盒（MiLaboratory LLC），结合带有唯一分子标识符（unique molecular identifiers, UMI）的5'端快速扩增cDNA末端（5'-RACE）技术构建cDNA文库。文库在Illumina NextSeq 500测序平台上，采用300循环测序试剂盒，以150+150 nt的双端测序模式完成测序。利用MIGEC（UMI阈值设为2）、MIXCR及VDJTOOLS软件从测序读段中推断得到克隆型。