Single-cell transcriptomic analysis of the adult mouse spinal cord reveals fundamental molecular diversity of autonomic and skeletal motor neurons

To characterize the diversity of cholinergic neurons in the spinal cord, we performed single-nucleus RNA sequencing on cholinergic-enriched pools of nuclei from the adult mouse. Overall design: Enrichment: Nuclei were enriched by fluorescence-activated nuclei sorting (FANS) of GFP+ cholinergic nuclei from reporter mice. Barcoding: Nuclei were loaded into a 10X Chromium device and barcoded via oligo-dT reverse transcription using the V3 chemistry. Library prep: Libraries were prepared by PCR and size-exclusion according to published 10X protocols. Sequencing: Libraries were sequenced using NextSeq 550 high-throughput, 150-cycle kits and NovaSeq 100-cycle kits.

为解析脊髓内胆碱能神经元的多样性，我们针对成年小鼠来源的胆碱能富集细胞核群开展了单细胞核RNA测序（single-nucleus RNA sequencing）。
实验整体设计如下：
- 细胞核富集：通过荧光激活细胞核分选（fluorescence-activated nuclei sorting, FANS）技术，从报告基因小鼠中分离GFP阳性的胆碱能细胞核，完成细胞核富集。
- 条形码标记：将细胞核加载至10X Chromium系统，采用寡聚dT（oligo-dT）反转录结合V3化学试剂体系进行条形码标记。
- 文库制备：参照已发表的10X官方实验流程，通过聚合酶链式反应（PCR）与尺寸排阻法完成文库构建。
- 测序实验：使用NextSeq 550高通量150循环测序试剂盒与NovaSeq 100循环测序试剂盒完成文库测序。