特异性投射神经元的单细胞分离与测序

Single-cell isolation and sequence of specific projecting neurons

目的:开发一种适用于特异性投射神经元转录组测序的单细胞分离方法。方法:用逆行示踪腺相关病毒(AAV)特异性标记成年小鼠丘脑底核(STN)投射至丘脑前核(ANT)的神经元,通过梯度离心将其制备成单细胞悬液,利用显微操纵器逐个吸取被荧光标记的神经元,运用Smart-seq2构建基因文库并进行质检和测序。结果:利用该方案成功分离到STN投射至ANT神经元;所构建的cDNA文库质量满足测序要求;通过测序结果比对,在目标神经元内检测到病毒表达的荧光蛋白基因。结论:本研究建立了一种适用于转录组测序的单细胞分离方法,可高效、高质量分离特异性投射神经元,为未来在靶细胞数目稀少的组织样本中进行转录组学研究提供了一种新的方案。

Objective:To develop a single-cell isolation and extraction method for transcriptome sequencing of specific projection neurons.Methods:Retrograde adeno-associated viruses(AAV) were used to specifically label subthalamic nucleus neurons(STN) that project to the anterior thalamic nucleus(ANT) of adult mice.STN neurons were then made into single-cell suspension through gradient centrifugation.Using a micromanipulator,the fluorescein-labeled neurons were aspirated.After the gene library construction,the Smart-seq2 technique was used for quality assessment and sequencing.Results:The STN-ANT projecting neurons were successfully extracted;the constructed cDNA library met the sequencing requirements.Furthermore,through sequence alignment,the fluorescent protein genes of the virus were successfully detected in the targeted neurons.Conclusion:The present study established a novel single-cell isolation and extraction method suitable for transcriptome sequencing,which can extract specific projection neurons with high efficiency and high quality.The study provided a new strategy to obtain cells from tissue with sparse number of target cells for future transcriptome research.