GSM8615374: mch_female; Mus musculus; RNA-Seq
After decapitation, the brain was removed, and the hypothalami of MCH-Cre; lsl-ZsGreen mice were dissected and frozen in liquid nitrogen immediately. On single nuclei preparation day, individual hypothalami tissue were lysed in 1ml of ice-cold EZ PREP buffer (D8938, Sigma). The tissue was homogenized with a glass dounce, twice at 250 rpm and 12 times at 1000 rpm and incubated on ice for 5 min. Nuclei were centrifuged at 500 g, 5 min after addition of 4 mL of nuclear lysis buffer and incubated on ice for 5 min. The supernatant was discarded, and the pellet was resuspended in 4 mL nucleus suspension buffer (NSB, 1x PBS, 0.01% BSA, and 0.1% RNase inhibitor (352235, Clontech). Nuclei were centrifuged at 500 g for 2 min. The supernatant was discarded and isolated nuclei were resuspended in 300 μl NSB and then filtered through a 30 μm cell strainer into sorting tubes for flow cytometry sorting on a BD FACSAria IIIu. Nuclei suspension was stained with DAPI (1:1000, 62248, Thermo Fisher Scientific) as nuclear marker. ZsGreen positive nuclei + DAPI positive nuclei were collected as MCH neuron nuclei. A FACSAria IIIu was used for gating and sorting nuclei. Single nuclei were identified based on side scatter (SSC-A) and DAPI fluorescence (area versus width). ZsGreen fluorescence was detected with 488 nm excitation and 530 nm emission. Sequencing libraries were generated using 10x Genomics Chromium Single-Cell 3′ Reagent kit v3 according to the standardized protocol. Paired-end sequencing was performed using an Illumina NovaSeq 6000.