title: GSM9328856: ZNF865 upregulated degenerative NP cells (32 Y M) 1; Homo sapiens; ATAC-seq
library_construction_protocol: Primary patient samples were prepared in technical duplicate, where 50,000 cells per replicate were taken from cryogenically frozen vials and centrifuged at 500 x g for 5 minutes at 4°C. The supernatant was aspirated, and the resulting cell pellet was washed with 250 µL of ice-cold 1X PBS, pH 7.4 (Thermo Scientific; 10010072). The cells were then centrifuged at 500 x g for 5 minutes at 4°C, the supernatant was aspirated, and the resulting cell pellet was resuspended in 50 µL of lysis buffer containing the following: 10 mM Tris-HCl, pH 7.4 (Sigma-Aldrich; T2194), 3 mM MgCl2 (Sigma-Aldrich; M1028), 10 mM NaCl (Sigma-Aldrich; 71386), 0.1% NP-40 (Millipore Sigma; 492016), 0.1% Tween-20 (Bio-Rad; 1610781), and 0.01% digitonin (Invitrogen; BN20061). Samples were then lysed via incubation on ice for 15 minutes and centrifuged at 500 x g for 5 minutes at 4°C.  Following lysis, 1 mL of wash buffer (10 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 10 mM NaCl, and 0.1% Tween-20) was added to each sample, which was then centrifuged at 500 x g for 5 minutes at 4°C. The supernatant was aspirated, and the resulting cell pellet was resuspended a second time in 1 mL of wash buffer. To pellet nuclei, cells were centrifuged at 500 x g for 10 minutes at 4°C. The supernatant was aspirated, and each resulting nuclei pellet was resuspended in transposition buffer containing the following: 10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 10% Dimethylformamide (Fisher Bioreagents; BP1160), and 1 µL of TDE1 Tagment DNA enzyme per sample (Illumina; 20034197) diluted in PBS, pH 7.4. For the transposition reaction, nuclei were incubated at 37°C for 30 minutes in an Eppendorf ThermoMixer C (EP5382000015) set to rotate at 1000 RPM. Following transposition, DNA was purified using the Zymo DNA Clean & Concentrator-5 kit (D4014) according to the manufacturer's instructions with a binding buffer to sample ratio of 5:1. The purified DNA was then amplified by PCR using a final concentration of 1X Phusion Master Mix (New England Biolabs; M0530), and 200 nM each of forward and reverse barcode primers. Amplified libraries were purified using a 1:1 volume ratio of AMPure XP beads (Beckman Coulter; A63882) and fragment size distribution and quality were assessed using a High-Sensitivity D1000 ScreenTape assay.