SPATIaL-seq of primary human T Follicular Helper (TFH) cells and naive CD4-positive helper T cells from tonsils of healthy volunteers. SPATIaL-seq of primary human T Follicular Helper (TFH) cells and naive CD4-positive helper T cells from tonsils of healthy volunteers

Genome Wide Association Studies (GWAS) have been successful in yielding >60 loci for Systemic Lupus Erythematosus (SLE). However, it is known that GWAS just reports genomic signals and not necessarily the precise localization of culprit genes, with eQTL efforts only able to infer causality to a minority of such loci. Thus, we sought to carry out physical and direct ‘variant to gene mapping’ by integrating results from high-throughput chromatin conformation capture and ATAC-seq assays. This experiment refers to the chromatin conformation capture part of our work. Detecting contacts between distant regions of the genome offers a powerful opportunity to understand GWAS signals that principally reside in non-coding regions, and thus likely act as regulatory elements for neighboring genes. To move beyond analyzing one locus at a time and to improve on the low resolution of available Hi-C data, we developed a massively parallel, high resolution Capture-C based method to simultaneously characterize the genome-wide interactions of all human promoters in any cell type. We refer to this method as genome-Scale Promoter-focused Analysis of chromaTIn Looping (SPATIaL-seq). We applied this approach to study the promoter ‘interactome’ of primary human T Follicular Helper (TFH) cells from tonsils of healthy volunteers (3 biological replicates), a model relevant to SLE as TFH operate upstream of the activation of pathogenic autoantibody-producing B cells during the disease. We also analyzed the promoter interactome of naive CD4-positive helper T cells (3 biological replicates). We designed a custom Agilent SureSelect library targeting both ends of DpnII restriction fragments that overlap promoters of protein-coding, noncoding, antisense, snRNA, miRNA, snoRNA and lincRNA transcripts. Each library was sequenced on 8 lanes of an Illumina HiSeq 4000.

全基因组关联分析（Genome Wide Association Studies, GWAS）已成功鉴定出超过60个与系统性红斑狼疮（Systemic Lupus Erythematosus, SLE）相关的基因座。然而，众所周知，GWAS仅能报告基因组信号，未必能精确定位致病基因；且表达数量性状位点（expression Quantitative Trait Loci, eQTL）研究仅能推断少数此类基因座的因果关联。因此，本研究尝试通过整合高通量染色质构象捕获与转座酶可及性测序（ATAC-seq）的实验结果，开展直接的“变异-基因定位”分析。本实验即为本研究中染色质构象捕获部分的工作。

检测基因组远距离区域间的染色质相互作用，为解析主要位于非编码区的GWAS信号提供了强有力的途径——此类信号大概率作为邻近基因的调控元件发挥功能。为突破单次仅分析单个基因座的局限，并改善现有Hi-C数据分辨率不足的问题，我们开发了一种大规模并行、高分辨率的基于Capture-C的方法，可在任意细胞类型中同时表征人类所有启动子的全基因组相互作用。我们将该方法命名为全基因组规模启动子聚焦染色质环分析（SPATIaL-seq）。

我们将该方法应用于研究来自健康志愿者扁桃体的原代人滤泡辅助性T（T Follicular Helper, TFH）细胞的启动子“相互作用组”（共3个生物学重复）；TFH细胞在疾病过程中位于致病性自身抗体产生B细胞活化的上游，因此该模型与SLE发病机制相关。我们同时分析了初始CD4阳性辅助T细胞的启动子相互作用组（共3个生物学重复）。

我们设计了定制化安捷伦SureSelect文库，靶向覆盖与蛋白编码、非编码、反义、小核RNA（small nuclear RNA, snRNA）、微小RNA（microRNA, miRNA）、小核仁RNA（small nucleolar RNA, snoRNA）以及长链非编码RNA（long intergenic non-coding RNA, lincRNA）转录本启动子重叠的DpnII限制性酶切片段的两端。每个文库均在Illumina HiSeq 4000的8个测序泳道上完成测序。