GSM7412362: e15_Het_1; Mus musculus; OTHER
E12.5 and E15.5 embryos were removed and placed in ice-cold carbogenated ACSF. Tails were clipped for PCR genotyping. During genotyping, embryonic brains were harvested and MGEs were dissected from each embryo and stored in ice-cold carbogenated ACSF. For E12.5, the entire MGE was removed as previously described92. For E15.5, the MGE was dissected under a fluorescent dissecting microscope to ensure collection of only Tom+ MGE cells and minimize collection of postmitotic MGE-derived cells in the striatum anlage and other ventral forebrain structures. Upon obtaining genotyping results (~90 minutes), MGEs from embryos of the same genotype were combined (when applicable) to generate single nuclei dissociations. MGEs were transferred to a 1 mL Dounce homogenizer containing DNA Lysis Buffer (10 mM Tris-HCl (Ph.7.4), 10 mM NaCl, 3 mM CaCl2, 0.1% Tween-20, 1.5% BSA and 0.1% IGEPAL CA-630 in nuclease-free water, 1 mL per sample). Lyse cells by douncing with pestle A and pestle B, ~10-15 times each. Place a 40 μm filter onto a pre-chilled 50 mL conical tube on ice and wet with 1 mL of DNA Nuclei Wash Buffer (in mM: 10 Tris-HCl (Ph.7.4), 10 NaCl, 3 CaCl2, with 0.1% Tween-20 and 1.5% BSA in nuclease-free water, 5 mL per sample). Transfer lysed nuclei suspension through pre-wetted filter, then rinse dounce with 1 mL Nuclei Wash Buffer and transfer through filter. Divide nuclei suspension into 2 pre-chilled 2 mL tubes and spin at 500 g for 5 minutes at 4oC. After removing supernatant, wash nuclei pellet once with 1 mL Nuclei Wash Buffer and then once with 1 mL of 1X CUT&Tag Wash Buffer (from Active Motif CUT&Tag IT Assay kit). Remove CUT&Tag Wash Buffer, leaving ~30-50 μL in each, triturate and combine nuclei suspensions from both tubes. Nuclei concentration was determined on Countess II FL Automated Cell Counter. 100,000-125,000 nuclei were used for each CUT&Tag reaction. Proceed with CUT&Tag IT Assay Kit (Active Motif, #53610) per manufacturer's instructions. Primary antibody was H3K27me3 (Cell Signaling, 9733T, 1:50) or H3K27ac (Cell Signaling, and secondary antibody was guinea pig anti-rabbit (Active Motif, 105465 from CUT&TagIT kit, 1:100). DNA quantity was determined with a Thermo Qubit and library quality characterized with an Agilent Tapestation. Libraries were balanced for DNA content and pooled before performing a final SPRIselect bead 1x left size selection and paired-end sequenced (50 x 50 bp) on an Illumina NovaSeq.