Patch-Seq of synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment

The goals of this study is to compare transcriptome profiles (RNA-seq) of synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment.Tg(HuC:H2B-GCaMP6f) fish of 4-5 weeks old were were fixed in a recording chamber containing extracellular fluid, and neurons with synchronized or non-synchronized calcium activity after CAS treatment were identified by two-photon imaging and collected separately with glass pipette. Single cells were transferred to the lysis buffer solution. Total RNA was extracted using TRIzol (Invitrogen) and then purified on RNeasy columns (Qiagen). Total RNA quality was assessed on a bioanalyzer (Thermofisher). RNA-Seq libraries (N=10) for each group were prepared using the Illumina TruSeq Strand mRNA Prep Kit according to the manufacturer's instructions. RNA-Seq libraries were sequenced using Illumina NextSeq 500, generating 75 bp paired-end reads for each sample. RNA-Seq reads untrimmed. Differential expression analysis was performed using the CPM (counts per million) function in the Bioconductor package edgeR (v 3.14.0). Low expression genes were excluded to make a simple correction for gene counts. Genes with P-value (instead of adjusted P-value) < 0.05 were assigned as differentially expressed. We identified differentially expressed genes (DEGs) between synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment. Synchronized neurons expressed much higher level of glutamate transporter genes (slc17a7a, slc17a6a), while non-synchronized neurons showed significantly higher expression of gad1b, suggesting that synchronized neurons are primarily glutamatergic, while non-synchronized neurons are mainly GABAergic. Overall design: To distinguish neurons with synchronized or non-synchronized calcium activity after CAS treatment, Tg(HuC:H2B-GCaMP6f) fish of 4-5 weeks old were fixed in a recording chamber containing extracellular fluid and subjected to two-photon imaging, and neurons were collected separately with glass pipette. RNA-Seq libraries (N=10) for each group were prepared using the Illumina TruSeq Strand mRNA Prep Kit according to the manufacturer's instructions.

本研究旨在比较经CAS处理后，斑马鱼背侧大脑皮层中同步化与非同步化神经元的转录组谱（RNA-seq）。选取4-5周龄的Tg(HuC:H2B-GCaMP6f)转基因鱼，将其固定于含细胞外液的记录槽中，通过双光子成像鉴定经CAS处理后钙活动同步化与非同步化的神经元，并用玻璃微管分别收集两类神经元。将单个细胞转移至裂解液中，使用TRIzol (Invitrogen)提取总RNA，随后通过RNeasy层析柱(Qiagen)进行纯化；总RNA质量通过生物分析仪(Thermofisher)进行评估。每组（N=10）构建RNA-seq文库，所用试剂盒为Illumina TruSeq Strand mRNA Prep Kit，操作严格遵循厂商说明书；使用Illumina NextSeq 500平台对文库进行测序，生成75 bp双端读段，原始测序读段未经过修剪。采用Bioconductor包edgeR(v3.14.0)中的CPM（每百万读段计数）函数进行差异表达分析，通过剔除低表达基因以对基因计数进行简易校正；以未校正P值<0.05作为差异表达基因(DEGs)的筛选标准。本研究最终鉴定出经CAS处理后斑马鱼背侧大脑皮层内同步化与非同步化神经元间的差异表达基因(DEGs)：同步化神经元的谷氨酸转运体基因(slc17a7a、slc17a6a)表达水平显著更高，而非同步化神经元则显著高表达gad1b，提示同步化神经元主要为谷氨酸能神经元，而非同步化神经元以GABA能神经元为主。整体实验设计：为区分经CAS处理后钙活动同步化与非同步化的神经元，选取4-5周龄的Tg(HuC:H2B-GCaMP6f)转基因鱼固定于含细胞外液的记录槽中，经双光子成像后通过玻璃微管分别收集神经元；每组（N=10）采用Illumina TruSeq Strand mRNA Prep Kit构建RNA-seq文库，操作遵循厂商说明书。