GSM7139703: Foxp1/2-cKO #1; Mus musculus; ATAC-seq
Striatal tissue for P9 mice was collected following rapid decapitation and brains were removed and placed in ice-cold artificial cerebrospinal fluid (ACSF; 126 mM NaCl, 20 mM NaHCO3, 20 mM D-Glucose, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2) bubbled with 95% O2 and 5% CO2. 500uM coronal sections were made in ACSF using a VF-200 Compresstome (Precisionary Instruments) and transferred to another chamber filled with ACSF. From 3 striatal sections, 6 punches of striatal tissue were collected (2 per hemisphere), flash frozen, and stored in -80C. Nuclei isolation for snATAC-Seq was done as follows: tissue was homogenized in a glass Dounce tissue grinder (25 times with pestle A, 25 times with pestle B; Sigma, Cat#D8938) in 1mL ice-cold EZ Nuclei Lysis Buffer (Sigma, Cat #NUC-101). Samples were incubated on ice for 3 minutes with an additional 1mL ice-cold EZ lysis buffer and then centrifuged at 500 X g for 5 minutes at 4C. Nuclei were then washed in 500uL Nuclei Wash Buffer (20uL of 1M Tris pH 7.4, 4uL of 5M NaCl, 6uL 1M MgCl2, 0.01% BSA, 20uL of 10% Tween-20 + 1.75mL H2O) and spun at 500 X g for 5 minutes again. The supernatant was then discarded, the pellet was resuspended in 150uL nuclei wash buffer, and filtered through FLOWMI tip strainer (Bel-Art Products, Cat# H13680-0040); more wash bufer was added as needed to help samples go through the FLOWMI tip. An aliquot was stained with Trypan Blue and counted under a microscope to determine concentration (targeting 4,000 nuclei/uL). Nuclei were then diluted to desired concentration, using 20X Nuclei buffer provided by 10X. Library was performed according to the manufacter's instructions (single cell ATAC v1.1 and v2 protocol, 10x Genomics). Isolated nuclei were mixed with the transposition mix which was followed by transposition. Then, 10X  master mix was and samples were loaded onto chip alongside  partitioning oil and gel beads. This results in generation of gel bead-in-emulsion (GEMs).  GEMs were then cleaned with Silane DynaBeads  followed by size-sleion using SPRIselect beads. Samples were cleaned with 80% ethanol  as well. Finally, libraries were constructed and  paired-end constructs with P5 and P7 sequences and a sample index were added during these final steps.