| Experiment Id | GSE224407 | Name | Single-Nucleus RNA Sequencing of Developing and Mature Superior Colliculus Identifies Neuronal Diversity and Candidate Mediators of Circuit Assembly |
| Experiment Type | RNA-Seq | Study Type | Baseline |
| Source | GEO | Curation Date | 2024-06-19 |
| description | The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates inputs from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment and refinement of neural connectivity. Here we performed single nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell-types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We used these data to identify Pax7 as a marker for an anatomically homogeneous population of GABAergic neurons. Lastly, we report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known for regulating axon guidance and synapse formation. Our data provide a valuable resource for interrogating the mechanisms of circuit development, and identifying markers for manipulating specific neuronal populations and circuits in the SC. Superior colliculus was microdissected from embryonic day 19 (E10), post-natal day 4 (P4), P8, or P21 C57BL/6J mice. Tissue was pooled from multiple animals and homogenized inRNAase-free lysis buffer (0.32 M sucrose, 3 mM CaCl2, 3 mM MgAc2, 0.1 mM EDTA, 10 mM Tris-HCl,1 mM DTT, 0.1% Triton X-100 in DEPC-treated water) using glass dounce homogenizer on ice. The homogenate was loaded into a polycarbonate ultracentrifuge tube containing sucrose solution (1.8 M sucrose, 3 mM MgAc2, 1 mM DTT, 10 mM Tris-HCl in DEPC-treated water) in the bottom and centrifuged at 107,000 g for 2.5 hours at 4°C. Supernatant was aspirated, and the nuclei containing pellet was incubated in RNAse-free 1x PBS, 0.04% BSA, 0.2 U/µl RNAse Inhibitor on ice before resuspending the pellet. The nuclear suspension was filtered twice through a 30 µm cell strainer RNAase-free. Single nucleus suspension was processed and sequenced using 10X Genomics 3' v3 snRNA-seq platform with a target capture of 2000 nuclei per sample. Libraries were sequenced on NovaSeq SP 100 (200,000 reads/nucleus). After sequencing, Illumina output was processed using CellRanger v3.0.2. Base call files for each sample were demultiplexed. A pre-mRNA reference was generated with cellranger mkref using the mm10 mouse genome. Each sample was aligned to the custom mm10 mouse reference genome using CellRanger. |
