Standard ATAC-seq on frozen samples

Single nuclei were isolated from frozen tissue with Dounce homogenization by following the nuclei isolation protocol in Omni-ATAC (Corces et al. 2017). In brief, green-bean-size frozen tissue was incubated in 800 µL of ice-cold 1× homogenization unstable buffer (5 mM CaCl2 [Alfa Aesar J63122], 3 mM Mg(Ac)2 [Sigma-Aldrich M5661], 10 mM Tris at pH 7.8 [Invitrogen 15568-025], 0.01667 mM PMSF [Sigma-Aldrich P7626], 0.1667 mM β-mercaptoethanol [Sigma-Aldrich M-6250], 320 mM sucrose [Sigma-Aldrich 84097-250], 0.1 mM EDTA [Invitrogen AM9290G], 0.1% IGEPAL CA-630 [Sigma-Aldrich 13021-50]) for 5 min on ice. Tissue was homogenized through 10 strokes with a loose pestle and 20 strokes with a tight pestle, and then 400 µL of the homogenized sample was mixed with 400 µL of 50% OptiPrep density gradient medium (Sigma-Aldrich D1556-250) to make a final concentration of 25% of OptiPrep density gradient medium (Sigma-Aldrich D1556-250) with homogenized tissue. After preparation of tissue mixture, a fresh 2-mL low-binding vial was taken and layers of 35% of OptiPrep density gradient medium (Sigma-Aldrich D1556-250), 29% of OptiPrep density gradient medium (Sigma-Aldrich D1556-250), and 25% of OptiPrep density gradient medium (Sigma-Aldrich D1556-250), mixed with the sample, were on the top of each other. The layered vial was centrifuged at 3000g for 20 min at 4°C. After gradient centrifugation, the top 1300 µL was discarded, and the 200 µL of the nuclei region was carefully collected in a fresh vial. Then 800 µL of ice-cold PBS was added and centrifuged at 500g for 10 min, followed by resuspension in 500 µL of ice-cold PBS. Fifty thousand nuclei were used for each reaction and prepared library by using standard ATAC protocol as stated in the section of standard ATAC-seq on FFPE samples (Buenrostro et al. 2013). The components for the solutions are as follows:

6× homogenization buffer stable master mix—30 mM CaCl₂ (Alfa Aesar J63122), 18 mM Mg(Ac)2, 60 mM Tris-HCl (pH 7.8; Invitrogen 15568-025);

6× homogenization buffer unstable solution—6× homogenization buffer stable master mix, 0.1 mM PMSF, 1 mM β-mercaptoethanol (Sigma-Aldrich M-6250);

1× homogenization buffer unstable solution—1× homogenization buffer stable master mix, 320 mM Sucrose (Sigma-Aldrich 84097-250), 0.1 mM EDTA (Invitrogen AM9290G), 0.1% IGEPAL CA-360 (Sigma-Aldrich 13021-50);

50% OptiPrep density gradient medium (Sigma-Aldrich D1556-250) solution—1× homogenization buffer stable master mix, 50% OptiPrep density gradient medium (Sigma-Aldrich D1556-250) solution;

29% OptiPrep density gradient medium (Sigma-Aldrich D1556-250) solution—1× homogenization buffer stable master mix, 160 mM sucrose, 29% OptiPrep density gradient medium (Sigma-Aldrich D1556-250) solution;

35% OptiPrep density gradient medium (Sigma-Aldrich D1556-250) solution—1× homogenization buffer stable master mix, 160 mM sucrose (Sigma-Aldrich 84097-250), 35% OptiPrep density gradient medium (Sigma-Aldrich D1556-250) solution.

The ATAC-seq libraries were sequenced on Illumina NovaSeq 6000, and at least 20 million 150-bp paired-end sequencing reads were generated for each library.