ATAC-Seq library prepared from DV1846 sample
Frozen mitral valve tissues were mechanically dissociated in liquid nitrogen using mortar and pestle. Grinded tissue was transferred into 15 ml Falcon tubes containing 5 ml of cold homogenization buffer (HB): 10 mM Tris-HCl pH 7.6, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 0.01% Digitonin, 250mM Sucrose, and EDTA-free complete protease inhibitors (Roche, Pleasanton, CA). The homogenate was filtered through 400 m cell strainer (pluriStrainer, Leipzig, Germany). Then, the isolation of nuclei was performed as described in Omni-ATAC protocol (Corces et al., 2017) with modifications. Specifically, after Dounce homogenization, the tissue homogenate was pelleted at 500g for 10 minutes at 4C. Tissue homogenate (700-800 l) was transferred to a 14 ml round bottom tube (Greiner Bio-One, Frickenhausen, Germany) and mixed with an equal volume of 50% iodixanol in HB to obtain a final 25% iodixanol. 1200 l of 30% iodixanol in HB was layered underneath the 25% mixture, and then 1200 l of 40% iodixanol in HB was layered below the 30% iodixanol. Nuclei were enriched at 3,000g for 20 minutes at 4C in a swinging-bucket centrifuge. Nuclei that accumulated at the 30%/40% interface were collected into a 1.5 ml Lo-Bind Eppendorf tube, stained with Trypan Blue, and manually counted under microscope. Library was prepared according to the Omni-ATAC protocol (Corces et al., 2017)