ATAC-seq of mus musculus: double positive thymocytes
Thymi from 3-6 week-old mice were removed and small cortical incisions made prior to mechanical agitation with wide-bore glass pipettes in DMEM/F-12 (Gibco) to liberate thymocytes. Cells were stained with antibodies in T cell FACS buffer and live, lineage-negative (DAPI-, CD11b-, CD11c-, CD19-, Gr-1-, Ter119-, NK1.1-, TCR-) gated cells were sorted as DP (CD4+, CD8+, TCRblo, CD69-), CD4SP (CD4+, CD8-, TCRbhi), and CD8SP (CD4-, CD8+, TCRbhi).   ATAC-seq was performed as described previously (Buenrostro et al., Nature Methods, 2013) with minor modifications. For nuclei isolation, 50,000-60,000 FACS-sorted cells were lysed in Thymocyte Hypotonic Buffer (25 mM HEPES, 5 mM MgCl2, 0.05 mM EDTA, 10% Glycerol, 0.1% NP40. Tagmentation reactions were performed for 30 minutes at 37 C using reagents from Illumina Tagment DNA TDE1 Enzyme and Buffer Kit. Tagmented DNA samples were purified with DNA Clean & Concentrator-5 kit (Zymo Research). Libraries were quantified by qPCR, amplified accordingly, and purified using DNA Clean & Concentrator kit. Paired-end, dual-index sequencing was performed on the Illumina NextSeq500 platform.