Eski Sema Elif, Dubois Christine, Singh Sumeet Pal
IRIBHM, Université Libre de Bruxelles (ULB).
Medecine Faculty, Université Libre de Bruxelles (ULB).
J Vis Exp. 2020 Jun 24(160). doi: 10.3791/61471.
High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.
高通量转录组和表观基因组分析需要制备单细胞或单细胞核悬液。制备含有完整细胞或细胞核的悬液涉及解离和通透步骤，这些步骤可能会引入不必要的噪音和不良损伤。特别是，某些细胞类型，如神经元，很难解离成单个细胞。此外，细胞膜通透以释放细胞核需要通过反复试验进行优化，这可能耗时、费力且在经济上不可行。为了提高高通量测序样品制备的稳健性和可重复性，我们描述了一种基于柱的快速无酶和无洗涤剂的细胞核分离方法。该方案能够在20分钟内从整个斑马鱼大脑中高效分离出细胞核。分离出的细胞核显示出完整的核形态且聚集倾向低。此外，流式细胞术可用于细胞核富集和清除细胞碎片，以便进行下游应用。该方案适用于软组织和培养细胞，为样品制备提供了一种简单且可操作的方法，可用于高通量分析，简化了成功进行单核RNA测序和ATAC测序实验所需的步骤。
