ATAC-seq library preparation

ATAC-seq was performed following a protocol from the Kaestner Lab (https://www.med.upenn.edu/kaestnerlab/assets/user-content/documents/ATAC-seq%20Protocol%20(Omni)%20-%20Kaestner%20Lab.pdf) with minor modifications. A total of 100,000 Kc cells were washed with 50 μL cold 1X PBS. Cell pellet was lysed with 50 μL cold lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 0.1% NP40, 0.1% Tween-20, 0.01% Digitonin) and incubated for 10 min on ice. Then 500 μL of wash buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20) was added to lysate. Nuclei were then collected by centrifuging at 500 × g for 10 min at 4 °C and nuclei were resuspended in 50 μL of transposition reaction mix [25 μL 2X TD buffer (Illumina), 16.5 μL PBS, 0.5 μL 10% Tween-20, 0.5 μL, 0.5 μL 1% Digitonin, 2.5 uL Tn5 enzyme (Illumina), 5 μL nuclease-free water] and incubated for 45 min at 37 °C at 1000 rpm (Eppendorf Thermomixer) for fragmentation. DNA was purified with Qiagen Minelute columns, and libraries were amplified by adding 10 μL DNA to 25 μL of NEBNext HiFi 2x PCR mix (New England Biolabs) and 2.5 μL of 25 μM each of Ad1 and Ad2 primers using 11 PCR cycles. Libraries were purified with 1.2× AMPure XP beads. All samples were sequenced with NextSeq-550 (Illumina) using 50 bp paired-end sequencing.