GSM9631010: Vehicle treated DVC, female, Epi Multiome chemistry, GEX, replicate 1; Mus musculus; RNA-Seq
Hsd11b2-IRES-Cre mice aged 5-10 weeks received bilateral stereotaxic injections of Cre-dependent AAV-H2b-mCherry into the NTS. After 2 weeks of recovery, mice were sacrificed between 9:00 am – 12:00 pm by rapid decapitation immediately after removal from the home cage. Brains were extracted and chilled in DMEM/F12 media slush (Thermo Fisher Scientific). Brains were then placed dorsal side up in a chilled stainless steel brain matrix (Stoelting, Wood Dale, IL) and 1 mm coronal sections of the caudal medulla were collected. Tissue from the Dorsal Vagal Complex was microdissected under a fluorescent stereoscope, frozen on dry ice, and pooled according to sex and treatment (vehicle or aldosterone). All pooled samples were stored dry at -80°C. Each sample underwent nuclear dissociation and purification procedures before being incubated with NucBlue (Thermo fisher scientific). After incubation, a portion of the nuclei were added to a collection tube with ATAC Buffer B (10X Genomics). Subsequently, mCherry+ nuclei were flow sorted into the same collection tube using a FACS Aria cell sorter with a 70 μm nozzle to remove poor quality nuclei and count the number of nuclei per sample. Pooled nuclei were immediately loaded on the 10x Chromium controller (10x Genomics) according to the manufacturer's protocol. Single Cell Multiome ATAC + Gene Expression was used to process all samples. cDNA and gene expression libraries were generated according to the manufacturer's instructions (10x Genomics). Gene expression and ATAC libraries were sequenced on an Illumina NovaSeq 6000 sequencer with the following sequencing configurations: GEX - Read1 - 28, i7 = 10, i5 = 10, Read2 = 90, ATAC - Read1 = 50, i7 = 8, i5 = 24, Read2 = 49..