Fig. 6 | Tracking mitochondria in ALS motor axons

This item is part of the Figshare Project:
Early mitochondrial dysfunction revealed across FUS- and TARDBP-ALS at single cell resolution

From Data Availability Statement for the paper in Nature Communications entitled:
Single-cell RNA sequencing reveals early mitochondrial dysfunction unique to motor neurons shared across FUS- and TARDBP-ALS
"We have deposited all raw and processed RNA sequencing data generated in this study on the NCBI Gene Expression Omnibus (GEO) under the accession number GSE226482. The C9orf72-ALS bulk RNA sequencing data was retrieved directly from the authors of the study.
[Items under this Figshare Project contain:] "Scans of fluorescent western blots, raw imaging files from confocal microscopy, the analysis files from Opera Phenix, qPCR data sets, and Seahorse assay result files."
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[Item specific description:]
Motor neurons were generated from iPSC by differentation according to Nijssen et al, 2019. BioProtocol (PMID: 33654821). Embryoid bodies containing motor neuron progenitors were attached whole at day 9, followed by maturation during which axons grow radially outward.
From day 27 onwards and as indicated, mitochondria were labelled with TMRM for 30 min prior to live cell imaging to record time-lapses.
These files are images and time-lapse videos of mitochondrial movement in iPSC-derived motor axons based on TMRM-stained mitochondria. The file format is .czi, through the ZEN software (Zeiss). Zeiss recommends using ImageJ and the ImageJ-based Fiji software package.
For analysis of mitochondrial movement, individual mitochondria were traced in Fiji using the TrackMate 7 extension with the StarDist detector and a Sparse LAP tracker (as in ‘mitotracker.py’). The mitochondrial traces were then annotated and analysed in R (version 4.2.0) using our custom package ‘mitotrackR’ (https://github.com/schwi24/mitotrackR).
Information about the microscopy file format CZI can be found here: https://www.zeiss.com/microscopy/en/products/software/zeiss-zen/czi-image-file-format.html

本数据集隶属于Figshare项目：单细胞分辨率下揭示FUS与TARDBP相关肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis，ALS）的早期线粒体功能障碍

以下为发表于《Nature Communications》的论文数据可用性声明：
论文题为《单细胞RNA测序揭示FUS与TARDBP相关肌萎缩侧索硬化（ALS）共享的运动神经元特异性早期线粒体功能障碍》
“本研究产生的全部原始与处理后的RNA测序数据已上传至NCBI基因表达综合数据库（NCBI Gene Expression Omnibus，GEO），登录号为GSE226482。C9orf72相关肌萎缩侧索硬化（ALS）的批量RNA测序数据直接取自该研究作者。

【本Figshare项目包含以下数据集】：
荧光免疫印迹扫描图、共聚焦显微镜原始成像文件、Opera Phenix高内涵成像系统分析文件、实时荧光定量PCR（quantitative real-time polymerase chain reaction，qPCR）数据集以及Seahorse细胞能量代谢分析结果文件。

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【本数据集专属说明】：
运动神经元由诱导多能干细胞（induced pluripotent stem cell，iPSC）通过分化诱导生成，分化方案参照Nijssen等2019年发表于《BioProtocol》的研究（PMID: 33654821）。包含运动神经元祖细胞的拟胚体于第9天整体贴壁，随后进入成熟阶段，此过程中轴突呈放射状向外生长。

自第27天起，按实验要求，在活细胞成像前使用四甲基罗丹明甲酯（Tetramethylrhodamine Methyl Ester，TMRM）对线粒体进行30分钟染色，以记录线粒体运动的延时影像。

本数据集包含基于TMRM染色线粒体的iPSC源性运动神经元轴突内线粒体运动的成像文件与延时视频，文件格式为.czi，通过蔡司ZEN软件（Zeiss ZEN）生成。蔡司官方推荐使用ImageJ及基于ImageJ开发的Fiji软件包进行相关处理。

针对线粒体运动的分析流程为：首先在Fiji软件中通过TrackMate 7插件，结合StarDist检测器与Sparse LAP追踪器（详见'mitotracker.py'）对单个线粒体进行轨迹追踪。随后使用R语言（版本4.2.0）及自研工具包'mitotrackR'（代码仓库地址：https://github.com/schwi24/mitotrackR）对线粒体轨迹进行注释与分析。

关于CZI显微镜成像文件格式的详细信息可查阅：https://www.zeiss.com/microscopy/en/products/software/zeiss-zen/czi-image-file-format.html”