Single cell transcriptome analysis of proprioceptive muscle afferents. Single cell transcriptome analysis of proprioceptive muscle afferents

We report on the single cell RNA sequencing of genetically identified adult, neonatal, and embryonic proprioceptors. High depth sequencing data was acquired for all developmental time points using plate-seq. For adult proprioceptors, bioinformatics analysis identified five molecularly distinct subsets. At least two of these subsets have been validated to correspond to group Ia muscle spindle afferents and group Ib GTO afferents, respectively. Overall design: Proprioceptors were labeled using intersectional genetic tools and fluorescently labeled neurons were isolated using fluorescence activated cell sorting (FACS). Neurons were directly deposited in lysis buffer in 96-well plates and subjected to RNA isolation, cDNA generation, library preparation and single cell sequencing. Single proprioceptor transcriptomes were filtered for high quality cells (>2000 UMI's), and limited satellite cell contamination (<10% Apoe).

本研究报道了经遗传学鉴定的成年、新生及胚胎本体感受器的单细胞RNA测序（single cell RNA sequencing）相关数据。本研究采用板测序（plate-seq）技术，为所有发育时间节点的样本获取了高深度测序数据。针对成年本体感受器，通过生物信息学分析鉴定出5个分子特征各异的亚型，其中至少两个亚型经验证分别对应Ia类肌梭传入神经元与Ib类高尔基腱器官（Golgi Tendon Organ, GTO）传入神经元。
实验整体设计：本体感受器通过交集遗传学工具完成标记，随后利用荧光激活细胞分选（fluorescence activated cell sorting, FACS）分离得到荧光标记的神经元。将神经元直接接种于96孔板的裂解缓冲液中，随后依次进行RNA提取、cDNA合成、文库制备及单细胞测序。对单个本体感受器转录组进行质量过滤，筛选出UMI计数大于2000的高质量细胞，且卫星细胞污染率受控（Apoe基因占比<10%）。