ATAC-seq of mus musculus: CD4 activated T cells
Spleen and lymph nodes from 3-6 week-old mice were isolated in RPMI 1640 (Gibco) supplemented with 10% FCS and 1% Penicillin-Streptomycin (10% RPMI). Cells were liberated by mincing with syringe plunger and filtered through 40 m strainer. After ACK lysis, cells were stained with antibodies in T cell FACS buffer. Live, lineage-negative (DAPI-, CD11b-, CD11c-, CD19-, Gr-1-, Ter119-, NK1.1-, TCR-) T cells (TCRbhi) were sorted as CD4+ Nave (CD8-, CD4+, CD44lo, CD62Lhi). For in vitro T cell activation, 96-well tissue-culture-treated microtiter plates (Corning) were coated with 1 g/ml anti-CD3 (clone 145-2C11) and 2 g/ml anti-CD28 (clone 37.51) and incubated at 37 C for 90 min. After 3 cold PBS washes, 200k FACS-sorted CD4+ Nave T cells were added to each well in 10% RPMI and incubated for 72 h at 37C, 5% CO2. Live (DAPI-), activated T cells were FACS sorted as CD4+ ACT.   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 ATAC lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20). Tagmentation reactions were performed for 30 minutes at 37C 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.