title: RNA-seq of FOXP3+ CD4+ regulatory T cells and their FOXP3- CD4+ conventional counterparts from human peripheral blood
design: RNA-seq of FOXP3+ CD4+ regulatory T cells and their FOXP3- CD4+ conventional counterparts from human peripheral blood
library_construction_protocol: Thymocytes were isolated by Ficoll-Hypaque (GE Healthcare) from the cell suspensions obtained by thymic tissue manual dispersion. CD4SP Tregs and Tconvs were sort-purified based on the surface expression of CD4, CD8, CD27, CD25 and CD127 in FACS Aria III (BD Biosciences), reaching purities always above 95%. CD3 was intentionally not used to avoid possible signalling, but the sorting strategy was always further validated in parallel using CD3 and intracellular Foxp3 in a Fortessa flow cytometer (BD Biosciences). The staining protocols have been previously described (Nunes-Cabaço et al., 2010). Thymic samples were obtained during paediatric reconstructive cardiac surgery, using tissue that would be otherwise discarded from children without evidence of immunodeficiency or syndromatic diseases. Conversion between annotations was made with “biomaRt”.  RNA samples were extracted from cell pellets of sorted 600,000 tTregs and tTconvs, using the AllPrep DNA/RNA kit (QIAGEN) and following the manufacturer's instructions.  Libraries were built selecting for polyadenylated RNA after depleting ribosomal fraction.