ATAC-seq of mus musculus: Bcl11b WT DN3 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. Bcl11b-cKO thymi were digested with 1.25 g/mL Collagenase IV (Sigma) and 1.0 g/mL DNase I (Sigma) in DMEM/F-12 for 5 min at 37C for efficient liberation of thymocytes. Cells were initially enriched via negative selection against CD8a using MACS with CD8a (Ly-2) microbeads (Miltenyi Biotec). Live, lineage-negative (DAPI-, CD11b-, CD11c-, CD19-, Gr-1-, Ter119-, NK1.1-, TCR-) DN (CD4- CD8-) cells were sorted as DN1 (CD25-, c-Kit+), DN2a (CD25+, c-Kit+), and DN3 (CD25+, c-Kit-).  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.