Guevara Elaine E, Erbaba Begün, Cresswell Gregory M, Edler Melissa K, Sherwood Chet C
Department of Evolutionary Anthropology, Duke University, Durham, NC, USA.
Department of Anthropology and Center for the Advanced Study of Human Paleobiology, The George Washington University, Washington DC, USA.
Yale J Biol Med. 2025 Jun 30;98(2):135-145. doi: 10.59249/LLUJ2834. eCollection 2025 Jun.
Epigenetic alterations are cell type-specific and require methods like single cell sequencing and cell type sorting by flow cytometry. These methods often rely on the availability of fresh tissue, yet postmortem frozen tissue is typically the only material available from non-experimental subjects, including humans and other nonhuman primates (NHP). Many insights can be gained from analysis of these precious samples. To this end, we developed a protocol for isolating intact nuclei from small starting amounts of postmortem frozen chimpanzee () cerebral cortex tissue. Isolated nuclei can be input directly into single cell epigenomics protocols like ATAC-seq or can be immunostained for enrichment of neuronal nuclei via fluorescent-activated nuclei sorting (FANS) followed by bulk epigenetic methods like methylome sequencing. We adapted and optimized this protocol based on existing human brain tissue protocols. Our protocol specifically addresses challenges presented by postmortem frozen NHP brain tissue, including high levels of myelin debris and reduced RNA integrity. We include key steps and troubleshooting guidance to improve nuclei quality and sorting outcomes, and we also discuss limitations and considerations for researchers interested in using these methods.
表观遗传改变具有细胞类型特异性，需要诸如单细胞测序和流式细胞术细胞类型分选等方法。这些方法通常依赖于新鲜组织的可用性，然而死后冷冻组织通常是从非实验对象（包括人类和其他非人类灵长类动物（NHP））获得的唯一材料。对这些珍贵样本的分析可以获得许多见解。为此，我们开发了一种从少量死后冷冻的黑猩猩大脑皮质组织中分离完整细胞核的方案。分离出的细胞核可以直接输入到诸如ATAC-seq等单细胞表观基因组学方案中，或者可以通过荧光激活细胞核分选（FANS）对神经元细胞核进行免疫染色富集，然后进行诸如甲基化组测序等批量表观遗传方法。我们基于现有的人类脑组织方案对该方案进行了调整和优化。我们的方案特别解决了死后冷冻的NHP脑组织带来的挑战，包括高水平的髓磷脂碎片和降低的RNA完整性。我们包括关键步骤和故障排除指南，以提高细胞核质量和分选结果，并且我们还讨论了对使用这些方法感兴趣的研究人员的局限性和注意事项。
