GSM7793667: D2 Foxp1cKO + Foxp1AAV #1 (mCherry); Mus musculus; RNA-Seq
We sacrificed P18 mice by rapid decapitation and quickly dissected the brain and transferred it into ice-cold nACSF that contained (mM): 125 NaCl, 25 NaHCO3, 10 Dextrose, 3 KCl, 1.25 NaH2PO4, 2 CaCl2.2H2O, 2 MgCl2 with 7.3-7.4 pH and 300-305 mOsm osmolality. We placed the brain onto an ice-cold 1.0 mm stainless steel brain matrix (Stoelting, Cat#51386) and obtained three 1.0 mm thick striatal sections which were then transferred into a petri-dish containing cold nACSF. Using forceps and under a dissection microscope, striatum was dissected out from these slices, flash frozen in liquid N2, and stored at -80°C. The striatal tissue was collected from P18 mice of required genotypes at different time points and stored. Protocol for isolating nuclei was adapted and modified from our previously published work6. Briefly, tissue was homogenized 20 times with pestle A, followed by 20 times with pestle B, in a glass Dounce tissue grinder (Sigma, Cat#D8938) in 2 ml ice-cold EZ Nuclei Lysis Buffer (Sigma, Cat #NUC-101). Homogenized samples were transferred into 15 ml tube with an additional 2 ml ice-cold EZ Nuclei Lysis Buffer, incubated on ice for 3 min and centrifuged at 500 X g for 5 min at 4°C. The supernatant was discarded, and nuclei pellet was resuspended in 3 ml ice-cold Nuclei Suspension Buffer (NSB; 1X PBS, 0.01% Ultrapure BSA, and 0.1% RNase inhibitor) and again centrifuged at 500 X g for 5 min at 4°C. The supernatant was then discarded, and nuclei pellet was resuspended in 300-400 µl NSB, and filtered through 40 µm size Flowmi cell strainer (Bel-Art Products, Cat# H13680-0040). An aliquot of the filtered nuclei suspension was mixed with Trypan Blue and transferred into a disposable cell counting chamber nuclei to visualize and count the healthy nuclei under a light microscope to determine desired nuclei concentration (targeting 700-1,200 nuclei/µl). Libraries from a total of 9 mice (3 mice/genotype) were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (10X Genomics, Cat#1000121) targeting 10,000 nuclei per sample as per the manufacturer's instructions. All mice were injected either with ControlAAV or Foxp1AAV in their striatum at P1 age where only a fraction of neurons was infected that were identified based on the presence of AAV transcripts. However, the proportion of AAV transcripts is exceptionally small in the pool of total transcripts that are captured by snRNA-seq experiment and therefore we used a modified approach 12 where we amplified the viral transcript specific sequences and prepared separate libraries. Briefly, we used 5% of the total cleaned up cDNA (amplified from the GEMs that were generated from each sample for the targeted 10,000 nuclei) and amplified the viral transcripts with reverse primer 10X_Enrich_R – 5'CTACACGACGCTCTTCCG3' and forward primers 10X_WPRE_F1 – 5'ACGAGTCGGATCTCCCTTT3' (for ControlAAV infected mice) and 10X_mCherry_F1 – 5'GCATGGACGAGCTGTACA3' (for Foxp1AAV infected mice) for 25 cycles using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, Cat# M0531S). We used different forward primers for ControlAAV and Foxp1AAV infected mice since ControlAAV vector has a WPRE sequence at the 3'end whereas we had to remove WPRE sequence from the AAVFoxp1 vector to accommodate Foxp1 and mCherry into the vector and thus have the mCherry sequence at 3'end of Foxp1AAV vector. The amplified PCR product was run on an agarose gel, where a ~250 bp product was excised from the gel and eluted using QIAquick Gel Extraction Kit (Qiagen, Cat#28706) in 20 µl elution buffer (NEB). 5% of this first round amplified product was used to perform a second round of amplification using the reverse primer, i. e. 10X_WPRE_F1 and forward primers 10X_WPRE_F2 – 5'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGAGTCGGATCTCCCTTT3' (for AAVControl infected mice) and 10X_mCherry_F2 –5'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTACAAGTAGACCCAGCTT3' (for AAVFoxp1 infected mice) for 20 cycles using the same Phusion polymerase. Once again, this amplified PCR product was loaded on an agarose gel where similar size product (~250 bp) was excised and eluted in 30 µl elution buffer. The eluted PCR products were once again cleaned up using SPRIselect Reagent and subjected to sample-index PCR amplification according to the manufacturer's instructions (10X Genomics) to generate cDNA libraries. Finally, both (total and AAV_transcript) libraries were subjected to quality control using 4200 TapeStation System (Agilent, Cat# G2991BA) and quantified using Qubit 2.0 Fluorometer (Invitrogen). For each sample, 5 nM of the total libraries and 0.5 nM of the AAV transcript libraries (10% of the total library) were pooled and sequenced on a NovaSeq 6000 (Illumina) platform on S4 flow cell.