GSM7404742: KO brain, mouse e12.5; Mus musculus; ATAC-seq
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 Multiome Lysis Buffer (in mM: 10 Tris-HCl (Ph.7.4), 10 NaCl, 3 CaCl2, 1 DTT, with 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 Multiome Nuclei Wash Buffer (in mM: 10 Tris-HCl (Ph.7.4), 10 NaCl, 3 CaCl2, 1 DTT, with 0.1% Tween-20, 1.5% BSA and 1 U/μL in nuclease-free water, 5 mL per sample). Transfer lysed nuclei suspension through pre-wetted filter, then rinse dounce with 1 mL Multiome 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 twice with 1 mL Multiome Wash Buffer and spin as above. Remove Multiome Wash Buffer, leaving ~20-30 μL of solution in each tube. Triturate solution to dissociate pellets, then combine nuclei suspensions into 1 tube. Nuclei concentration was determined on Countess II FL Automated Cell Counter. Nuclei suspensions were diluted to ~3,000-4,000 nuclei/μL, with 5 μL being used for 10x Genomics Multiome kit per manufacturer's instructions. E15.5 data: MGE from 4 WT, Het and KO embryos from 2 different E15.5 litters were combined to generate 1 biological rep. E12.5 data: MGE from 1 WT, Het and KO mouse from a single E12.5 litter were used to generate 1 biological rep. Total cell numbers that passed QC: E12.5 WT = 6,391; E12.5 Het = 6,608; E12.5 KO = 8,546; E15.5 WT = 11,477; E15.5 Het = 10,027; E15.5 KO = 8,607. Joint libraries for scRNA-seq and scATAC-seq were created using 10X Genomics Single Cell Multiome ATAC + Gene Expression kit (1000285) by following manufacturer's protocols.