GSM9631021: Aldosterone-treated DVC, GEM-X chemistry, replicate 3; 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 caudal medulla was placed in chilled RNAprotect solution (QIAGEN, Aarhus, Denmark) and then the Dorsal Vagal Complex was microdissected under a fluorescent stereoscope. During dissections, tissue was pooled according to treatment (vehicle or aldosterone). All pooled samples were incubated overnight at 4°C in RNAprotect®. RNAprotect solution was then removed from sample tubes the following day before storing samples at -80°C. Each sample underwent nuclear dissociation and purification procedures before being incubated with NucBlue (Thermo fisher scientific). After incubation, nuclei were flow sorted together into RT Reagent B (10x Genomics) using a Cytek Aurora CS cell sorter with a 70 μm nozzle to remove poor quality nuclei, enrich for mCherry positive nuclei, and count the number of nuclei per sample. Sorted nuclei were immediately loaded on the 10x Chromium controller (10x Genomics) according to the manufacturer's protocol. Single Cell 3' GEM-X chemistry was used to process all samples. cDNA and gene expression libraries were generated according to the manufacturer's instructions (10x Genomics). Gene expression libraries for 'Rep 2' were sequenced on an Illumina NovaSeq 6000 sequencer (Read1 - 28, i7 = 10, i5 = 10, Read2 = 90). Gene expression libraries for 'Rep 3' were sequenced on an AVITI24 (Element Biosciences) (Rep 2) sequencer using a 150-cycle kit with the following sequencing configuration: i7 = 10, i5 = 10, Read1 = 29, Read2 =91.