Purpose: White matter (WM) abnormalities are associated with various central nervous system (CNS) disorders, and the optic nerve provides a unique opportunity to study WM pathology. Large animal models offer a more suitable platform for preclinical testing of novel therapeutic strategies for optic neuropathy due to their similarities to humans in size and relevant anatomy. Transcriptomic analyses of optic nerve tissue are essential for understanding the underlying pathological mechanisms. However, extracting high-quality RNA from the optic nerve in large animals remains challenging. Methods: We utilized in situ hybridization and single-nucleus RNA sequencing (snRNA-seq) to examine mRNA expression in WM cells and gray matter (GM) cells. Results: We discovered that mRNA expression levels in WM cells were only 15% to 66% of those in GM neurons. To overcome the low mRNA yield, we developed a specialized RNA extraction protocol for the intra-canalicular optic nerve in large animal models, achieving an RNA integrity number (RIN) of 6.8 ± 0.06. For single-cell transcriptomics (scRNA-seq), we obtained a cell density of 1.0 × 105 cells/µL, cell viability of 93% ± 1.84%, and an agglomeration rate of 5.37% ± 0.75%. This approach is also applicable for postmortem human optic nerve with a RIN of 8.3 ± 0.3 using snRNA-seq. Conclusions: We first discovered that the mRNA expression in the WM was significantly lower than that in the GM. Our RNA extraction protocol from large animal models enhances transparency and reproducibility in transcriptomic studies of optic nerve and other WM tissues.