Nuclei ez prep buffer

After a section was taken for spatial transcriptomics, the remaining xenograft biopsy tissue was thawed from OCT and washed with PBS. Nuclei were subsequently isolated using Nuclei Lysis Buffer containing Nuclei Isolation Kit: Nuclei EZ Prep Buffer (Sigma) supplemented with cOmplete ULTRA Tablets (Sigma) and SUPERase IN (Thermo) and Promega RNAsin Plus nuclease inhibitors. Tissue was minced into <1 mm pieces in 2 mL of Nuclei Lysis Buffer. Samples were transferred to a Dounce homogenizer (Kimble) and homogenized. An additional 2 mL of Nuclei Lysis Buffer was added to the sample and incubated for 5 minutes on ice. Samples were passed through a 40 μm filter into a 50 mL conical tube. Samples were centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was removed and the pellet was washed with 4 mL of Nuclei Lysis Buffer containing 1% Bovine Serum Albumin for 5 minutes on ice. Samples were centrifuged at 500 × g for 5 minutes at 4°C. Samples were passed through a 5 μm filter into a 50 mL conical tube and then centrifuged again. Nuclei were resuspended in a solution containing phosphate-buffered saline (PBS), 1% BSA, and 0.1% RNAse inhibitor.

CUT&RUN was performed on the embryonic and adult prefrontal cortex as previously described (Skene and Henikoff 2017 (link); Brodie-Kommit et al. 2021 (link)). Three biological replicates were included for each age and genotype. Briefly, E14 mouse embryonic cortex or P60 mouse prefrontal cortex were dissected out, and nuclei were isolated using Nuclei EZ Prep Buffer (Sigma-Aldrich 4432370) and counted on cell cytometer. 300k nuclei were bound to the Concanavalin A-coated beads for each CUT&RUN reaction. Then, each aliquot of bead/nuclei were incubated with a primary antibody, including Rb-MYT1L (0.5 µg, Millipore ABE2915), Rb-H3K4me1 (1 µg, Abcam ab8895), Rb-H3K4me3 (1 µg, Active Motif 39159), Rb-H3K27ac (1 µg, Active Motif 39133), and Rb IgG (1 µg, Jackson ImmunoResearch 011-000-003), at 4°C on the nutator overnight. Next, to bind pAG-MNase fusion protein to the antibodies, beads were incubated with diluted CUTANA pAG-MNase (1:20, EpiCypher 15-1016) on the rotator at 4°C for 1 h. Chromatin digestion was performed at 0°C with the addition of CaCl₂ (100 mM) for 30 min. To digest the RNA and release the cleaved DNA fragments, reactions were incubated with Stop Buffer at 37°C for 30 min in the thermocycler. Magnetic stands were used to bind beads afterwards, and supernants containing DNA fragments were retrieved for sequencing library preparation.