GSM7912875: AgRP-Gq_POMC-Gi_CNO_1; Mus musculus; RNA-Seq
A portion of the hindbrain containing the AP and NTS was dissected in HibernateA Medium (A1247501) on ice and snap frozen on liquid nitrogen and  stored at -80°C overnight. Homogenates were produced using a Dounce homogeniser in 1 ml of homogenate buffer (100 µM of DTT, 0.1% Triton X-100, 2X EDTA-free Protease Inhibitor, 0.4 U/µl of RNasin RNase inhibitor, and 0.2 U/µl of Superase.In RNase Inhibitor in nuclei isolation medium [250 mM of sucrose, 25 mM of KCl, 5 mM of MgCl2, and 10 mM of Tris buffer at a pH of 8.0 in nuclease-free water with 1 µl/ml of DRAQ5 on ice. The homogenates were centrifuged at 900xg for 10 min at 4 C, and pellets were then resuspended in homogenate buffer with 25% Opti-Prep diluted in iodixanol dilution medium (250 mM of sucrose, 150 mM of KCl, 30 mM of MgCl2, and 60 mM of Tris buffer with a pH of 8 in nuclease-free water). Each suspension was layered on top of separate 29% OptiPrep solutions and centrifuged for 20 min at 13,500xg at 4 C. For each sample, the pellet was removed and resuspended in 500 µl of wash buffer (1% BSA, 0.4 U/µl of RNasin, and 0.2 of U/µl in PBS). The samples were passed through a 35 µm cell strainer. Fluorescent-activated cell sorting (FACS) was performed using a BD Influx cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). The gating was set according to size and granularity using FSC and SSC to capture singlets and remove debris, and fluorescence at 647/670 nm to detect DraQ5 staining. Each sample was sorted into separate tubes, each with a total of 20,000 particles/40ul. Sequencing libraries for the single-nuclei suspension samples were generated using 10X Genomics Chromium Single-Cell 3' Reagent kit according to the standardized protocol. RNA from lysed nuclei was reverse transcribed and cDNA was PCR amplified for 19 cycles.