single nucleus ATAC-seq of Homo sapiens: adult brain middle temporal gyrus: Sample H1826405D_A11
"We sorted single NeuN+ nuclei into 1.5 uL of Nextera Tn5 transposition reaction (0.6 uL Tn5 enzyme, 0.75 uL tagmentation buffer, 0.15 uL 1% IGEPAL CA-630) in Eppendorf semi-skirted 96-well plates. In the cases where glial cells were sorted, we first sorted neurons from the sample using PE-NeuN+ staining, and then treated with DAPI (1 ug/uL) for 1-2 minutes prior to sorting glial cells as DAPI+NeuN- events. Immediately following sorting we briefly centrifuged plates, vortexed, centrifuged plates again, and then incubated plates at 37oC for 30 minutes for transposition. After transposition we added 0.6 uL Proteinase K Cleanup Buffer (recipe below), vortexed briefly and centrifuged, and incubated at 40oC for an additional 30 minutes, then froze plates at -20 oC until library prep. For library prep, we purified tagmented DNA with 1.8x vol/vol Ampure XP beads (Beckman-Coulter catalog # A63881), eluted DNA in 11 uL and then PCR-amplified with Nextera Index kit primers (#FC-121-1012 from Illumina) using KAPA HiFi HotStart ReadyMix (KAPA Biosystems #KK2602) in a 30 uL reaction (72o 3:00, 95o 1:00, cycle 22x [98o :20, 65o :15, 72o :15], 72o 1:00). We purified PCR products using 1.8x Ampure XP beads, and quantified libraries using Agilent BioAnalyzer High Sensitivity DNA Chips (catalog # 5067-4626). Then sample libraries were pooled evenly and sequenced with paired-end 50bp reads on Illumina NextSeq 500 machine using Mid Output chip (SeqMatic, Fremont CA USA)."