RNA-sequencing (RNA-seq) of induced pluripotent stem cells (iPSC) and iPSC-derived astrocytes from control and Parkinson's disease patients carrying LRRK2 G2019S point mutation

Purpose: The goal of this study is to compare the NGS-derived from transcriptome profiling (RNA-seq) of human iPSC, human iPSC-derived astrocytes from control and Parkinson's disease LRRK2 G2019S, and human commercial astrocytes to gain insight into the identity of human iPSC-derived astrocytes in vitro during the differentiation process. Total RNA was assayed for quantity and quality using Qubit® RNA HS Assay (Life Technologies) and RNA 6000 Nano Assay on a Bioanalyzer 2100. The RNASeq libraries were prepared from total RNA using the TruSeq®Stranded mRNA LT Sample Prep Kit (Illumina Inc., Rev.E, October 2013). Briefly, 500ng of total RNA was used as the input material and was enriched for the mRNA fraction using oligo-dT magnetic beads. The mRNA was fragmented in the presence of divalent metal cations and at high temperature (resulting RNA fragment size was 80-250nt, with the major peak at 130nt). The second strand cDNA synthesis was performed in the presence of dUTP instead of dTTP, this allowed to achieve the strand specificity. The blunt-ended double stranded cDNA was 3´adenylated and Illumina indexed adapters were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. The libraries were sequenced on HiSeq2000 (Illumina, Inc) in paired-end mode with a read length of 2x76bp using TruSeq SBS Kit v4. We generated over 30 million paired-end reads for each sample in a fraction of a sequencing v4 flow cell lane, following the manufacturer's protocol. Image analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.18.66.3) and followed by generation of fastq.gz sequence files by CASAVA. Results: We found that transcriptome of human iPSC-derived astrocytes is similar to human commercial astrocytes than human iPSC. Conclusion: These results suggest that iPSC-derived astrocytes resemble human commercial astrocytes validating the differentiating protocol used. Overall design: Human iPSC-derived astrocyte identity was obtained by comparing human iPSC from the same control and Parkinson's disease LRRK2 G2019S patients and human commercial astrocytes. mRNA profiles were generated by deep sequencing using Illumina HiSeq2000.

研究目的：本研究旨在对比源自人类诱导多能干细胞（induced pluripotent stem cell, iPSC）、来自对照个体及帕金森病LRRK2 G2019S突变患者的人类iPSC分化星形胶质细胞，以及商业购得的人类星形胶质细胞的下一代测序（next-generation sequencing, NGS）转录组谱（RNA测序，RNA-seq）结果，以深入解析体外分化过程中人类iPSC来源星形胶质细胞的特性。
总RNA的含量与质量采用Qubit® RNA HS定量试剂盒（Life Technologies公司）与Bioanalyzer 2100的RNA 6000 Nano试剂盒进行检测。
RNA测序文库以总RNA为起始材料，使用TruSeq®链特异性mRNA LT样本制备试剂盒（Illumina公司，Rev.E版，2013年10月）构建。具体步骤简述如下：取500ng总RNA作为起始样本，利用寡聚dT磁珠富集mRNA组分；在二价金属阳离子与高温条件下将mRNA片段化，所得RNA片段大小为80~250nt，主峰位于130nt；以dUTP替代dTTP进行第二链cDNA合成，以此实现链特异性；将平端双链cDNA进行3´端腺苷酸化，并连接Illumina索引接头；通过15轮PCR富集连接产物，最终利用Agilent 2100 Bioanalyzer的DNA 7500试剂盒对完成的文库进行质量验证。
将文库置于HiSeq2000测序仪（Illumina公司）上，采用TruSeq SBS Kit v4试剂，以双端测序模式进行测序，读长为2×76bp。参照厂商操作流程，为每个样本生成了超过3000万条双端reads，占用单条v4测序流通槽的部分泳道。
本批次测序的图像分析、碱基识别与质量评分均通过厂商软件实时分析（Real Time Analysis, RTA 1.18.66.3）完成，随后通过CASAVA工具生成fastq.gz格式的测序文件。
研究结果：本研究发现，人类iPSC来源星形胶质细胞的转录组与商业购得的人类星形胶质细胞更为相似，而非人类iPSC。
研究结论：上述结果表明，iPSC分化获得的星形胶质细胞与商业购得的人类星形胶质细胞特性相近，验证了本研究采用的分化方案。
总体实验设计：通过对比来自同一对照个体及帕金森病LRRK2 G2019S突变患者的人类iPSC，以及商业购得的人类星形胶质细胞，解析人类iPSC来源星形胶质细胞的特性。本研究通过Illumina HiSeq2000进行深度测序，获取了mRNA表达谱。