title: GSM9220277: Astrocytes Donor A22 R1 ATAC; Homo sapiens; ATAC-seq
library_construction_protocol: (ATAC) Primary human astrocyte cultures were thawed at 37 oC in a water bath until a small ice crystal remained. Cells were immediately diluted in RPMI supplemented with 10% FBS and centrifuged at 300 x g for 5 minutes. Pellets were washed twice in PBS containing 0.04% BSA and passed through a 40 um cell strainer to remove debris. Cell viability and counts were assessed using a hemocytometer prior to nuclei isolation. For lysis, cells were resuspended in 100 uL of chilled lysis buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Non-idet P-40 substitute, 0.01% digitonin, 1% BSA, 1 mM DTT, and 1x protease inhibitors] and incubated on ice for 3 minutes. Nuclei were washed three times in 1 mL of wash buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1x protease inhibitors] at 500 x g for 5 minutes at 4 oC. Nuclei were resuspended in PBS + 0.04% BSA, counted, and typically yielded ~4 x 10^6 nuclei per sample. (ATAC) Fifty thousand nuclei were resuspended in 12.5 uL of 1x nuclei buffer and incubated with pA-Tn5 transposase preloaded with sequencing adapters. Tagmentation was performed in a thermocycler at 37 oC for 30 minutes. Tagmented DNA was purified using the Zymo DNA Clean & Concentrator kit (Zymo Research) and eluted in 21 uL of nuclease-free water. Libraries were amplified in 50 uL reactions using NEB-Next High-Fidelity 2x PCR Master Mix (NEB, Cat# M0541) and 2.5 uL each of 10 uM indexed i5 and i7 primers. PCR cycling conditions were: 72 oC for 5 min, 98 oC for 30 s, followed by 12 cycles of 98 oC for 10 s, 63 oC for 30 s, and 72 oC for 1 min, with a final extension at 72 oC for 1 min. Amplified libraries were cleaned with 0.75x SPRI-select beads (0.75x) with two 80% ethanol washes and eluted in 20 uL water.