GSM7139742: Foxp1/2-cKO_P56 #2; Mus musculus; RNA-Seq
P56 mice were sacrificed by rapid dissection and brains were quickly removed and placed in ice-cold 1X PBS. Using a brain matrix with 1mm markings, striatal sections were obtained, as determined by visual cues indicating the presence of  the striatum. Using forceps, the striatum was separated from the cortex, flash frozen, and stored in -80C. Nuclei isolation for snRNA-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 2mL ice-cold EZ Nuclei Lysis Buffer (Sigma, Cat #NUC-101). Samples were incubated on ice for 5 minutes with an additional 2mL ice-cold EZ lysis buffer and then centrifuged at 500 X g for 5 minutes at 4C, washed with 2mL ice-cold EZ lysis buffer, and incubated on ice for another 5 minutes before being spun again. Nuclei were then washed in 500uL Nuclei Suspension Buffer (NSB; 1X PBS, 0.01% UltraPure BSA, and 0.1% RNase inhibitor) and mixed with 900uL nuclei sucrose cushion buffer (Sigma, Cat #NUC-201). This mixture was layered on top of another 500uL of sucrose cushion buffer and spun at 13,000 X g for 45 minutes at 4C. The supernatant was then discarded, the pellet was resuspended in 60uL NSB, and filtered through FLOWMI tip strainer (Bel-Art Products, Cat# H13680-0040); more NSB 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 700-1,200 nuclei/uL). 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 were then isolated in a similar fashion as adults, with only one 5 minute centrifugation in EZ Nuclei Lysis buffer.  No sucrose cushion was used. Again, samples were filtered through the FLOWMI tip strainer, stained with Trypan Blue, counted under a microscope, and stored on ice until library generation. Library was performed according to the manufacter's instructions (single cell 3' v3 and v3.1 protocol, 10x Genomics). Isolated nuclei were mixed with the 10X  master mix and loaded with partitioning oil and gel beads onto the provided chip. This results in generation of gel bead-in-emulsion (GEMs).  GEMs were then retrotranscripted to cDNA; these contained  Ilumina R1 primer sequence, Unique Molecular Identifier (UMIs) and the 10x Barcode. Silane DynaBeads were used to purify cDNA which were then amplified by PCR. SPRIselect beads were used to select proper sized fragments. Samples were cleaned with 80% ethanol after every amplification step as well. Finally, libraries were constructed and an Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added during these final steps.