ATAC-seq Protocol for Epigenomic Profiling

ATAC-seq^{13 (link)} was performed by resuspending 1,000 to 20,000 FACS sorted cells in 50 μl nuclei lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630) and centrifuged at 500g for 30 min at 4°C. Next, the supernatant was removed and cells resuspended in 25 μl tagmentation mix (300 mM NaCl, 100 mM EDTA, 0.6% SDS, 1.6 μg Proteinase-K) and placed in a thermocycler at 37°C for 1 h. Cells were lysed and high molecular weight DNA removed using a 0.6× SPRI-bead negative selection and low molecular weight DNA purified by 1.2× SPRI-bead positive selection. The resulting tagmented DNA was PCR amplified and indexed using Nextera Indexing primers (Illumina, Inc) and 2× HiFi Hotstart Ready Mix (Roche, Inc). Following PCR, high molecular weight DNA was removed using a 0.3× SPRI-bead negative selection and low molecular weight DNA purified by 1× SPRI-bead positive selection. Final libraries were checked for ATAC-seq specific patterning on a Bioanlyzer and quantitated by qPCR prior to pooling at equimolar ratios and sequenced on a HiSeq2500 using 50bp PE chemistry.