GSM5284790: ATACseq_VTA1_DN; Mus musculus; ATAC-seq
For collection of single-nucleus ATAC-seq (snATAC-seq), male C57Bl/6Nl (RRID: IMSR_CR:027; WT) mice, ages 7 weeks, was sacrificed by cervical disolation. Brain was removed and the tissue containing the midbrain VTA was dissected from each hemisphere at room temperature and rapidly frozen on dry ice. Frozen midbrain was kept at -80C until further processing. 10x Genomics scATAC-seq library was generated from midbrain VTA according to the 10X Genomics manual “Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing Rev B” for flash frozen tissue with minor adjustments. Briefly, 500 uL 0.1X lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.01% Tween-20, 0.01% Nonidet P40 Substitute 0.001% Digitonin) was added to the frozen samples and immediately homogenized using a pellet pestle (15x), followed by 5 min incubation on ice. The lysate was pipette mixed 10x, then incubated 10 min on ice. Finally, 500 uL of chilled wash buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 2 mM MgCl2, 1% BSA, 0.1% Tween-20) was added to the lysed cells, and the suspension was passed through 30 uM CellTrics strainers (Th Geyer, cat# 7648779) The final ~500 l nuclei suspension was stained with DAPI (final concentration 0.03 ug/mL) for ~5 min.   Around 200,000 DAPI positive events were sorted using a BD FACSAria III Flow Cytometer with 70µm nozzle configuration with sample and sort collection device cooling set to 4°C into 300 l Diluted Nuclei buffer (commercial buffer from 10X Genomics) in a 1,5mL Eppendorf tube. A first gate excluded debris in a FSC/SSC-plot and a consecutive, second gate in a DAPI-A/DAPI-H-plot was used to exclude doublets and nuclei with incomplete DNA content. The collected nuclei were centrifuged at 500g for 5 min at 4°C and resuspended in 20µl Diluted Nuclei Buffer. The nuclei concentration was determined using a Countess II FL Automated Cell Counter in DAPI fluorescence mode. snATACseq libraries were prepared following the Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 User Guide. Briefly, nuclei were loaded on a microfluidics chip together with RT reagents, beads with oligo-dT tags and oil to create an emulsion. Afterwards the reverse transcription reaction takes place inside the droplets. The barcoded cDNA is recovered from the emulsion, amplified and cleaned using a bead purification process. The cDNA is then using for library construction including enzymatic fragmentation, adapter ligation and sample index PCR. Libraries were sequenced with either an Illumina NextSeq 500 machine using paired-end 75 bp reads (for VTA-1, 150 cycles), or by NovaSeq 6000 using paired-end 75 bp reads (for VTA-2, 100 cycles).