title: GSM8420492: WDR26 KO HeLa_biol rep 2 [ATAC]; Homo sapiens; ATAC-seq
library_construction_protocol: the cells were washed with ice-cold PBS, scraped, and transferred to a fresh microcentrifuge tube. The viability of the cells was then determined using Trypan blue and the TC20 Automatic Cell Counter; if > 95%, 2x105-cell aliquots were prepared and centrifuged at 500 x g for 5 minutes at 4°C. The supernatant was carefully removed and the resulting pellet was resuspended in 200 µL of ATAC Resuspension Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) supplemented with digitonin (100 units/mL [final]), Tween-20 (0.1% [final]), and NP-40 (0.1% [final]) and incubated on ice for 3 minutes. The reaction was then quenched by transferring the sample to 15 mL Falcon tubes containing 4 mL ATAC Resuspension Buffer supplemented only with Tween-20 (0.1% [final]) and centrifuged at 500 x g for 10 minutes at 4°C. The supernatant was removed, leaving behind a nuclei-containing pellet. The pellet was resuspended in 500 µL of 1X TD Buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 10 mM DMF, 35% PBS) supplemented with digitonin (100 u/mL [final]) and Tween-20 (0.1% [final]); afterwards, viability and nuclei concentration was determined using Trypan blue and the TC20 Automatic Cell Counter. If 0%, 2x105-cell aliquots were prepared in 190 µL of 1X TD Buffer. After 10 µL of 100 nM Tn5 enzyme is added to the sample, the tagmentation is performed at 37°C for 30 minutes, with shaking at 700 rpm. The DNA was then immediately isolated using the Zymo DNA clean and concentrator-5 (D4014). PCR was performed using the Illumina i5 and i7 indexing primers containing adaptors for 8 cycles (75°C, 5 min; 98°C, 30 s; 8 cycles of (98°C,10 s; 63°C, 30 s; 72°C, 60 s); 4°C indefinitely. The PCR amplified DNA was cleaned using the Zymo DNA clean and concentrator-5, double size selected using Ampure XP beads (Cat#: A63880) and quantitated using Collibri library quantitation kit (Cat#: A38524100).