Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
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DOI：
10.1186/s12864-019-5608-2
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发表时间：
2019-03-20
期刊：
影响因子：
4.4
通讯作者：
Durinck, Kaat
中科院分区：
文献类型：
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作者：
Loontiens, Siebe;Depestel, Lisa;Durinck, Kaat
背景在细胞类型特异性启动子的控制下表达荧光报告基因的转基因斑马鱼品系，使得能够对FACS分选的细胞群体进行转录组分析。RNA质量和产量是精确表达谱的关键决定因素。有限的细胞数量和FACS诱导的细胞应激使得分选的斑马鱼细胞的RNA分离是一个微妙的过程。我们的目的是优化的工作流程，以提取足够数量的高质量的RNA从有限数量的FACS分选细胞从Tg（fli1a：GFP）斑马鱼胚胎，这可用于准确的基因表达analysis.ResultsWe评估两个合适的RNA分离试剂盒（theRNAqueous微和RNeasy加微试剂盒），并确定分选细胞直接进入裂解缓冲液是成功的关键步骤。对于低细胞数，这确保了直接细胞裂解，保护RNA免于降解，并导致更高的RNA质量和产量。我们表明，这在具有分选细胞的裂解缓冲液的高达0.5倍稀释度下工作良好。在我们的分选设置中，RNAqueous micro kit和RNeasy plus micro kit分别对应30，000和75，000个细胞。分选更多的细胞会将裂解缓冲液稀释得太多，并且需要使用收集缓冲液。我们还证明，在RNA分离后需要额外的基因组DNA去除步骤，以完全清除任何污染基因组DNA中的RNA。对于cDNA合成和文库制备，我们将SmartSeq v4全长cDNA文库扩增、Nextera XT标签片段化和样品条形码化组合。使用这个工作流程，我们能够产生高度可重复的RNA测序results.ConclusionsThe提出的优化的工作流程，能够产生高品质的RNA，并允许准确的转录组分析的小种群的分选斑马鱼细胞。
BackgroundTransgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis.ResultsWe evaluated two suitable RNA isolation kits (theRNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5x dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results.ConclusionsThe presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.