bulk ATAC-seq of Homo sapiens: adult brain middle temporal gyrus: Sample 171010_L4NeuNpos
"For bulk ATAC-seq, we pelleted tissue, discarded supernatant, and replaced with 50-100 uL of Nextera DNA library reaction (#FC-121-1031 from Illumina) containing 0.1% IGEPAL-630 (NP-40 alternative), and then pipetted up and down vigorously 25-50 times using a P200 pipette, and then incubated at 37oC for one hour for transposition. We then added 1 mL of Homogenization Buffer (recipe below) to quench the reaction, pelleted samples at 1000g for 5 minutes at 4oC, resuspended samples in 1 mL fresh homogenization buffer, released nuclei from samples using ~10-15 strokes of a loose-fitting dounce pestle followed by ~10-15 strokes of a tight-fitting dounce pestle, then filtered nuclei with a 70 um nylon mesh strainer, and pelleted nuclei at 1,000xg for 10 minutes at 4oC. To stain, we resuspended nuclei in 500 uL of ice-cold Blocking Buffer (recipe below) containing PE-NeuN antibodyand 1 ug/mL 4'-diamino-phenylindazole (DAPI, MilliporeSigma catalog # D9542), rocked samples for 30 minutes at 4oC, then pelleted at 1,000xg for 5 minutes at 4oC, and finally resuspended samples in 500 uL fresh ice-cold blocking buffer before sorting nuclei on a FacsARIA III.   Using scatter profiles to eliminate debris and doublets, we sorted bulk samples as DAPI+NeuN+ from layers 1-6, or as DAPI+NeuN- from layer 1 and layer 5 samples, at 5,000-10,000 cells per sample, into 200 uL of blocking buffer in low-binding Eppendorf 1.5 mL tubes. We pelleted sorted nuclei at 1,000xg for 10 minutes at 4oC, followed by resuspension in 50 uL Proteinase K Cleanup Buffer and 37oC incubation for 30 minutes, and then freezing at -20oC until library prep and sequencing.  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 17x [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 NextSeq machines (SeqMatic, Fremont CA USA)."