Distinct waves from the hemogenic endothelium give rise to layered Lymphoid Tissue Inducer cell ontogeny [bulk RNA-seq]

LTi cells are part of the ILC family and essential for the formation of secondary lymph nodes within the embryo. This data set contains the analysis of lymphoid tissue (LTi) cell ontogeny, studied by expression profile analysis with RNA bulk sequencing of the populations associated with LTi ontogeny isolated from fetal liver vs. embryonic periphery at embryonic stage E13.5. They indicate a proliferating precursor population mainly in the fetal liver, while the embryonic periphery harbors the definitive lineage. E13.5 embryos from RORgammat-eGFP mice were digested for 20 min at 37C with Liberase/DNase . All subsequent procedures were at 0-4°C. Cells were enriched using anti-CD45 microbeads and an AutoMACS magnetic cell separator according to manufacturer's instructions and stained and sorted based on the presence of surface markers CD45, IL7Ralpha, alphabeta7, RORgammat, CD4. Lineage positive cells were excluded using F4/80, CD3e, CD19, CD8a, Ly6G. Cell viability was evaluated using LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Life Technologies). LTi cells and precursors were sorted using a FACSAria III. RNA was isolated using a RNA purification kit (Norgen Biotek Corp. 51800) and the quality and concentration of the RNA was determined with the bioanalyzer using RNA 6000 pico kit (Agilent 5067-1513) and Qubit fluorometer (ThermoFisher) using the Qubit dsDNA HS Assay Kit (ThermoFisher Q32854). Only those samples with a RIN value above 8 were used for subsequent library preparation. Samples were pre-amplified with the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech).淋巴组织诱导细胞（LTi cells）属于固有淋巴细胞（Innate Lymphoid Cell, ILC）家族，是胚胎体内次级淋巴结形成的必需组分。本数据集涵盖淋巴组织诱导（LTi）细胞发育谱系的相关分析：研究人员针对胚胎发育阶段E13.5时，从胎肝与胚胎外周组织中分离得到的、与LTi细胞发育相关的细胞群，开展了批量RNA测序（RNA bulk sequencing）表达谱分析。分析结果表明，增殖性前体细胞群主要富集于胎肝中，而胚胎外周组织则包含终末分化的特征性细胞谱系。实验流程如下：取自RORγt-eGFP小鼠的E13.5胚胎组织，经Liberase/脱氧核糖核酸酶（DNase）于37℃下消化20分钟；后续所有操作均在0-4℃环境中进行。细胞通过抗CD45磁珠结合AutoMACS磁性细胞分选仪，按照厂商说明书完成富集；随后依据表面标志物CD45、IL7Rα、αβ7、RORγt、CD4的表达情况进行染色与分选，并通过F4/80、CD3ε、CD19、CD8α、Ly6G排除谱系阳性细胞。细胞活性通过活死细胞固定染色试剂盒（LIVE/DEAD Fixable Blue Dead Cell Stain Kit，Life Technologies）进行评估。LTi细胞及其前体细胞通过FACSAria III流式细胞分选仪完成分选。RNA提取采用RNA纯化试剂盒（Norgen Biotek Corp. 51800），RNA的质量与浓度分别通过搭载RNA 6000 Pico试剂盒（Agilent 5067-1513）的生物分析仪，以及采用Qubit dsDNA HS检测试剂盒（ThermoFisher Q32854）的Qubit荧光定量仪进行测定。仅RNA完整性数（RIN值）高于8的样本，方可用于后续文库构建。样本采用SMART-Seq v4超低起始量RNA试剂盒（Clontech）进行预扩增。