GSM8729824: CFA, SRP-001, scATAC; Rattus norvegicus; ATAC-seq
After one hour, the rats were euthanized, and the periaqueductal gray (PAG) was dissected from their flash-frozen brains. To isolate cell nuclei from the midbrain PAG, the tissue was flash-frozen, dissected with a cryotome, minced into small pieces, homogenized, filtered through a 70 μm strainer, and centrifuged. ReadiDrop 7-AAD cell viability dye (Bio-Rad) was added, and live nuclei (7-AAD positive) were sorted using a BioRad Cell Sorter S3e in purity mode. Nuclei were counted with a Nexcelom automated Cellometer using acridine orange/propidium iodide (AOPI) staining, and the nuclei stock concentration was adjusted to target 10,000 nuclei per sample. The samples were bulk transposed, and approximately 16,100 nuclei were loaded into each channel of the microfluidic chip, with GEMs generated using the 10x Chromium controller and reagents from the Chromium Next GEM Single Cell ATAC Kit, 1,000,280 (10x Genomics), following the manufacturer's instructions After post-GEM cleanup with Dynabeads and SPRIselect (Beckman Coulter), samples underwent pre-amplification PCR and were then divided for constructing ATAC and GEX libraries. Quality control for the libraries was performed using an Agilent Bioanalyzer 2100 with High Sensitivity DNA chips. After verifying sample traces, each library was normalized based on the average fragment size, and concentration was determined using a Qubit 4.0 fluorometer (Thermo Fisher Scientific). The pooled libraries were subsequently sequenced using Illumina high output kits on a NovaSeq 6000 platform from Illumina