title: GSM8016997: VNO scRNA-seq male replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: C57BL/6J animals: 6 male, 10 female were euthanized, VNOs were dissected and processed separately. The sensory epithelium was separated using forceps and immediately placed in EBSS equilibrated with 95% CO2, 5% O2. Neuroepithelial tissue from multiple animals of same sex was transferred to the papain dissociation buffer(20U/mL Papain,1mM L-cysteine, 0.5mM EDTA, 12 U/mL RNase-free DNase). The tissue was cut into small pieces and the suspension was transferred to 14 mL tube. The suspension was incubated with gentle shaking at 37°C for 30 minutes on a thermomixer (Eppendorf) by passing 95% CO2 ,5% O2 on top of the liquid headspace. During this incubation, the solution was triturated with fire-polished Pasteur pipette every 10 minutes and at the end the incubation. After incubation, the suspension was passed sequentially through 70µm, 35µm filters to remove tissue debris. The filtered suspension was further layered over a mix of ovomucoid inhibitor (2.5 mg/mL) and albumin (2.5 mg/mL) in EBSS and centrifuged at 400 x g for 5 minutes at room temperature to remove subcellular debris or membrane fragments. The supernatant was discarded, and the resuspended cell pellet was used for library preparation after Hank's balanced Salt solution (HBSS) wash. Dissociated neurons resuspended in HBSS were used for library preparation. Using hemocytometer and trypan blue staining, cell density was estimated at 1200 cells/ul and 1100 cell/ul for male and female samples, respectively with greater than 80% cell viability. Single-cell capture, library preparation was done using a Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit v3.1 on a 10X Chromium controller (10X Genomics). Single cell suspension from male and female samples was loaded onto two separate wells of different chips giving a total of four libraries (2 male and 2 female samples).