Fast-ATAC Sequencing of Sorted Cells

This protocol has been optimized for blood cells. We note that digitonin is a gentle detergent and this protocol may not be ideal for cell lines and other cell types that are more resistant to lysis. 5,000 sorted cells in FACS Buffer were pelleted by centrifugation at 500 RCF for 5 minutes at 4C in a pre-cooled fixed-angle centrifuge. All supernatant was removed using two pipetting steps being careful to not disturb the not visible cell pellet. 50 ul transposase mixture (25 ul of 2x TD buffer, 2.5 ul of TDE1, 0.5 ul of 1% digitonin, 22 ul of nuclease-free water) (Cat# FC-121-1030, Illumina; Cat# G9441, Promega) was added to the cells and the pellet was disrupted by pipetting. Transposition reactions were incubated at 37°C for 30 minutes in an Eppendorf ThermoMixer with agitation at 300 RPM. Transposed DNA was purified using a QIAgen MinElute Reaction Cleanup kit (Cat# 28204) and purified DNA was eluted in 10 ul elution buffer (10 mM Tris-HCl, pH 8). Transposed fragments were amplified and purified as described previously^{48 (link)} with modified primers^{23 (link)}. Libraries were quantified using qPCR prior to sequencing. All Fast-ATAC libraries were sequenced using paired-end, dual-index sequencing on a NextSeq with 76×8×8×76 cycle reads.