Decoding Gene Networks Governing Hypothalamic and Prethalamic Neuron Development

Purpose: sc/snRNA-Seq and scATAC-Seq were performed to characterize the developing hypothalamus Cell Dissociation for scRNA-Seq: To prepare samples for scRNA-Seq, tissues were dissociated in Hibernate medium (Hibernate-E Minus Calcium (#HECA500, BrainBits) for embryos or Hibernate-A Minus Calcium (#HACA500, BrainBits) for postnatal stages), supplemented with 2 mg/ml papain (#LS003119, Worthington Biochemical) and 0.5 mM Glutamax. The enzyme mixture was pre-incubated for 15 minutes, then added to tissues for 5 - 30 minutes with 0.1 U/μl RNase inhibitor (N2615, Promega). Toward the end of digestion, 100 μg/ml final DNase I (#, 4716728001, Sigma-Aldrich) was added. Cells were subsequently centrifuged, resuspended in HABG buffer, filtered through a 40 μm cell strainer (BAH136800040-50EA, Sigma-Aldrich), and resuspended in HABG buffer containing RNase inhibitor (0.5 U/μl). Cell viability and counts were assessed with Trypan Blue staining (#15250061, Thermo Fisher Scientific) and a hemocytometer / Nuclei Preparation for scATAC-Seq:Nuclei for scATAC-Seq were extracted following post-cell dissociation and purified following a 10x Genomics protocol. Nuclei count and yield were verified with Trypan Blue staining and a hemocytometer / Nuclei Preparation for snRNA-Seq:For snRNA-Seq, adult mouse brains were rapidly dissected, and 100 um coronal sections were prepared using a mouse brain matrice. Regions spanning the ZI, TRN, and thalamus were microdissected under a dissection microscope. Nuclei were extracted following a 10x Genomics protocol.