title: Thymus GFP- Tregs replicate 1
design: RNA sequencing analysis Rag2-GFP+ and Rag2-GFP- CD4+CD8-Foxp3-Thy1.1+ Tregs from mouse thymus and spleen were sorted by FACS and total RNA was immediately extracted by using the RNeasy® Micro Kit (QIAGEN) (including a DNase I treatment step) and the quality of the RNA was assessed by using an Agilent 2100 BioAnalyzer (Agilent Technologies). Three biological replicates were performed per cell population. Libraries for RNA-sequencing (RNAseq) were prepared according to the TotalScript RNA-seq protocol (Epicentre), starting from 5 ng of high-quality total RNA (i.e. RIN>7) and using the Oligo(dT) primer synthesis strategy. The quality of each library was assessed by using an Agilent 2100 BioAnalyzer. Samples were indexed and sequenced on Illumina HiSeq 2000 (paired-end 2x100bp). Reads were trimmed by using Cutadapt (v1.3), removing low-quality bases (-q < 10) and clipping adapter sequences. High-quality RNAseq reads were aligned to the mouse reference genome mm10 by using TopHat (v2.0.5) (11). Mice Rag2-Gfp transgenic mice (Rag-GFP) (W. Yu et al., Nature 400, 682 (1999)) on a C57BL/6 (B6) genetic background were provided by Dr. Pamela Fink (T. E. Boursalian, J. Golob, D. M. Soper, C. J. Cooper, P. J. Fink, Nature Immunol 5, 418 (2004)), B6 Foxp3-Thy1.1 knock-in mice by Dr. Alexander Rudensky (A. Liston et al., Proc Natl Acad Sci U S A 105, 11903 (2008)), and B6 GK mice by Dr. Andrew Lew (Y. Zhan, A. J. Corbett, J. L. Brady, R. M. Sutherland, A. M. Lew, Xenotransplantation 7, 267 (2000)). 