title: GSM4498349: snRNA-seq avMLPA_5; Mus musculus; RNA-Seq
library_construction_protocol: The brains were extracted and sectioned into 300 μm coronal sections. Sections containing the MPA were microdissected to isolate the avMLPA.  The dissected avMLPA was dissected and placed into a Dounce with homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitors). The sample was homogenized using a tight pestle with 10 strokes. IGEPAL solution (5%, Sigma) was added to a final concentration of 0.32%, and 5 additional strokes were performed. The homogenate was filtered through a 40-μm filter, and OptiPrep (Sigma) was added to a final concentration of 25% iodixanol. The sample was layered onto an iodixanol gradient and centrifuged at 10,000g for 18 minutes as previously described. Nuclei were collected between the 30% and 40% iodixanol layers and diluted to 80,000-100,000 nuclei/mL for encapsulation. All buffers contained 0.15% RNasin Plus RNase Inhibitor (Promega) and 0.04% BSA. Single nuclei were captured and barcoded for whole-transcriptome libraries using the 10X Genomics Chromium v2 platform according to the manufacturer's recommendations, collecting one library of approximately 10,000 nuclei from each of the 5 animals. Libraries were processed according to manufacturer's (10X Genomics) protocols. The cDNA was amplified, fragmented and adapters were added for sequencing on a Nextseq 500 benchtop DNA sequencer (Illumina).     For enrichment of virally-derived transcripts, a fraction (1 μL) of the non-fragmented cDNA was PCR amplified. The forward primer (5'-GCATGGACGAGCTGTACA) was designed to anneal to the sequence uniquely present at the 3' terminus of mCherry. The reverse primer (5'-CTACACGACGCTCTTCCG) was designed to anneal to the R1 sequence, uniquely present at the 5' terminus, to the 10X barcode and UMI sequence which were introduced during the reverse transcription. The result of the PCR is preferential amplification of the viral-derived transcripts, while simultaneously retaining the cell-barcode sequence necessary to assign each viral transcript to a particular cell/nucleus. Following PCR amplification (18 cycles, Hot Start High-Fidelity Q5 polymerase, NEB # M0494S), the product was purified using 0.6X SPRI select reagent (Thermo Fisher Scientific # NC0406406) and 1ul of the 50ul eluent used in a second PCR reaction. The forward primer for the second PCR   (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgtaaggcgcgccataac) was designed to anneal to the sequence uniquely present between the mCherry and loxP sites of the the AAV-DIO-Gq-DREADD-mCherry vector. In addition, this primer introduced the R2 sequence necessary for later library amplification. The reverse primer (5'-CTACACGACGCTCTTCCG) was the same as in the first PCR reaction. The result of the PCR is again preferential amplification of the viral-derived transcripts, while simultaneously retaining the cell-barcode sequence necessary to assign each viral transcript to a particular cell/nucleus. Following PCR amplification (18 cycles, Hot Start High-Fidelity Q5 polymerase, NEB # M0494S), all the libraries were indexed according to the 10X protocol, pooled, and sequenced on a Nextseq 500 benchtop DNA sequencer (Illumina). 10X snRNA-seq
attributes.tag: GEO Accession
attributes.value: GSM4498349