A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design.
A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design.
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DOI：
10.1093/nar/gkw1242
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发表时间：
2017-04-20
影响因子：
14.9
通讯作者：
Waldmann R
中科院分区：
文献类型：
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作者：
Arguel MJ;LeBrigand K;Paquet A;Ruiz García S;Zaragosi LE;Barbry P;Waldmann R
单细胞 RNA 测序方法有助于研究细胞间的变异性。 5΄ 选择性转录组分析方法允许同时定义转录起始大小，并且比仅提供接近 3΄ 末端的内部序列的 3΄ 选择性方法具有优势。目前唯一存在的 5΄ 选择性方法需要在 cDNA 扩增后进行昂贵且劳动密集型的片段化和细胞条形码编码。我们开发了优化的 5΄ 选择性工作流程，其中所有细胞索引均在片段化之前完成。通过我们的方案，可以在 Fluidigm C1 微流体装置中进行细胞索引，从而显着降低成本和劳动力。我们还设计了优化的独特分子标识符，显示出更少的序列偏差和测序错误的脆弱性，从而提高了分子计数的准确性。我们为 Illumina 和 Ion Proton 测序仪提供全面的实验工作流程，使单细胞测序的成本范围与 qPCR 分析相当。
Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.