Single-nuclei sequencing of Parvalbumin interneurons in mouse visual cortex after optoTrkB activation

After deep anesthesia by pentobarbital, the mice were perfused with ice-cold NMDG ACSF, and the brains were isolated and sliced by vibratome in NMDG ACSF. Then the slices were exposed by LED blue light, and incubated in a modified HEPES aCSF for one hour. The visual cortex was Isolated from the slices under microscope with red light, and tissues were homogenized, and nuclei were extracted. All procedures were performed with RNase free in the dark condition. The nuclei were stained with Hoechst 33342 and mouse monoclonal anti-NeuN antibody (Sigma-Aldrich, MAB377), and double positive nuclei were sorted by FACS. Then the sorted nuclei were used to make cDNA Library with Chromium Single Cell 3' Gene Expression v3 library preparation kit (10x Genomics), and sequenced with NovaSeq 6000 (Illumina). Data was pre-processed using CellRanger 3.0, and analysed by Chipster version 3.16/Seurat v3. Overall design: snRNA-seq of neurons in the visual cortex

经戊巴比妥（pentobarbital）深度麻醉后，对小鼠灌注冰冷NMDG人工脑脊液（NMDG ACSF），随后取出大脑并在NMDG ACSF中使用振动切片机进行脑片切片。将脑片置于LED蓝光下照射后，于改良HEPES人工脑脊液（HEPES aCSF）中孵育1小时。随后在红光显微镜下从脑片中分离视觉皮层组织，将组织匀浆并提取细胞核。所有操作均在无RNase（核糖核酸酶）的避光条件下进行。细胞核经赫斯特33342（Hoechst 33342）及小鼠抗NeuN单克隆抗体（Sigma-Aldrich，货号MAB377）染色后，通过荧光激活细胞分选术（FACS）分选得到双阳性细胞核。分选得到的细胞核使用Chromium单细胞3'基因表达文库制备试剂盒v3（10x Genomics）构建cDNA文库，并通过NovaSeq 6000测序平台（Illumina）完成测序。测序数据采用CellRanger 3.0进行预处理，并通过Chipster 3.16版本/Seurat v3进行分析。实验整体设计：视觉皮层神经元的单细胞核RNA测序（snRNA-seq）。