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. Overall design: E13.5 embryos from ROR?t-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, IL7Ra, a4ß7, ROR?t, 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).