GSM6043651
Nuclei were isolated by dissociating 20 mg of fresh brain or liver tissue in 400 µl of lysis buffer (1M Tris-HCl, 1M MgCl2, 1M NaCl, 10% Nonidet-P40) by passing through a polished silanized Pasteur pipette. Samples were left to incubate at 4˚C for 5 min before repeated dissociation. Following an additional 5 min incubation, samples were treated with 2.5 ml of Wash buffer (1x PBS, 1% BSA), dissociated and filtered through a 30 µm cell strainer. Samples were centrifuged at 500 g for 5 min at 4˚C, with the remaining pellet resuspended in 110 µl Wash buffer. To estimate nuclei recovery efficiency, 10 µl of nuclei solution was combined with 10 µl of DAPI solution (1:5,000 in PBS) and loaded on a hematocytometer. The remaining nuclei suspension was treated with 180 µl of 1.8 M sucrose solution (2 M sucrose cushion solution (S9308-475ML), Nuclei PURE Sucrose Cushion Buffer (S9058-120ML)) and homogenized. The 280 µl of nuclei suspension was gently layered onto 1 ml of 1.3 M sucrose solution (S9308-475ML, Nuclei PURE Sucrose Cushion Buffer (S9058-120ML) and centrifuged at 3,000 g for 10 min. Nuclei pellets were suspended in 100 µl of wash buffer. Assays for transposase-accessible chromatin using sequencing (ATAC-Seq) were performed on nuclei samples containing 50,000 nuclei. Isolated nuclei were centrifuged at 500 g for 10 min at 4˚C. The pellet was suspended in transposition reaction mix (Illumina Tagment DNA TDE1 Enzyme and Buffer Kit) and incubated at 37˚C for 30 min on a thermomixer (Eppendorf) at 1,000 rpm. DNA was then isolated with Zymo DNA Clean & Concentrator-5 Kit (Integrated Sciences) and DNA elutes were amplified by PCR with 25 µM of indexing primers. Library purification was performed by isolating amplified DNA with SPRIselect beads (Beckman Coulter Life Sciences) with an ethanol wash before suspension in nuclease-free water.