E13.5 embryos from RORγt-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, IL7Rα, α4β7, RORγt, 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). Pre-amplification of 8 samples with the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech). Libraries were generated with samples' barcode and molecular indexing, the latter being used in order to provide an absolute digital measurement of genes expression levels.
GEO Accession
GSM4647910