Glyoxal as alternative fixative for single cell transcriptome experiments. Glyoxal as alternative fixative for single cell transcriptome experiments

Within just a few years single cell RNA sequencing has become a frequently used standard method in many research fWithin just a few years single cell RNA sequencing has become a frequently used standard method in many research facilities worldwide. Declining sequencing costs and further refinements of the available protocols will pave the way for previously unimaginable prospects, up to single cell transcriptomic maps of entire organisms. The sample collection process can constitute a severe bottleneck in scRNA-seq experiments especially for solid tissues. Lengthy dissociation protocols are not uncommon to obtain sufficient amounts of starting material and can lead to significant changes in the gene expression profiles of cells. Preservation of the transcriptome prior to single cell dissociation can overcome this setback. Here we present an extensive performance analysis of glyoxal as an alternative fixative for scRNA-seq application. High numbers of transcripts and genes were recovered from glyoxal-fixed cells subjected to Drop-seq methodology with the best performance in Drosophila cells. While glyoxal fixation in Drosophila Kc167 and human HEK 293T cells revealed transcriptome data similar to the unfixed condition, reduced library complexity was observed for the human sample. We found the integrity of Drosophila intestinal tissue maintained following glyoxal fixation, while dissociation of the fixed tissue allowed sufficient cell isolation. In conclusion, we present glyoxal as a well-suited fixative for Drosophila samples that allows high-quality single cell transcriptomic analysis and successful intestinal tissue disaggregationcilities worldwide. Declining sequencing costs and further refinements of the available protocols will pave the way for previously unimaginable prospects, up to single cell transcriptomic maps of entire organisms. The sample collection process can constitute a severe bottleneck in scRNA-seq experiments especially for solid tissues. Lengthy dissociation protocols are not uncommon to obtain sufficient amounts of starting material and can lead to significant changes in the gene expression profiles of cells. Preservation of the transcriptome prior to single cell dissociation can overcome this setback. Here we present an extensive performance analysis of glyoxal as an alternative fixative for scRNA-seq application. High numbers of transcripts and genes were recovered from glyoxal-fixed cells subjected to Drop-seq methodology with the best performance in Drosophila cells. While glyoxal fixation in Drosophila and human cells revealed transcriptome data similar to the unfixed condition, reduced library complexity was observed for the human sample and might require further protocol optimization. We found the integrity of Drosophila intestinal tissue maintained following glyoxal fixation, while dissociation of the fixed tissue allowed sufficient cell isolation. In conclusion, we present glyoxal as a well-suited fixative for Drosophila samples that allows high-quality single cell transcriptomic analysis and successful intestinal tissue disaggregation. Overall design: Experiments were performed in two biological replicates, with technical PCR replicates included in the first experiment (DS27) and Dropseq run replicates in the second experiment (DS34). In total, 7 libraries have been generated and analysed. *** Raw data could not be provided due to institutional restrictions concerning human sequence data ***

仅在短短数年内，单细胞RNA测序（single cell RNA sequencing）已成为全球众多研究机构中常用的标准实验方法。测序成本的持续下降与现有实验方案的进一步优化，将为此前难以想象的研究前景铺平道路，乃至实现完整生物体的单细胞转录组图谱构建。在scRNA-seq实验中，样本采集流程往往是严重的瓶颈，针对实体组织样本尤为如此。为获取足够量的起始材料，冗长的组织解离方案十分常见，而这可能会导致细胞基因表达谱发生显著改变。在单细胞解离前对转录组进行保存，便可攻克这一难题。本研究针对乙二醛（glyoxal）作为scRNA-seq应用的替代固定剂开展了全面的性能分析。通过Drop-seq技术对经乙二醛固定的细胞进行检测，可回收大量转录本与基因，其中在果蝇（Drosophila）细胞中表现最佳。果蝇Kc167细胞与人类HEK 293T细胞经乙二醛固定后，其转录组数据与未固定组相似，但人类样本的文库复杂度出现下降，可能需要进一步优化实验方案。我们发现，经乙二醛固定后果蝇肠道组织的完整性得以维持，且对固定后的组织进行解离可获得足够量的分离细胞。综上，乙二醛是适配果蝇样本的优质固定剂，可支持高质量的单细胞转录组分析，并实现肠道组织的有效解离。整体实验设计：实验设置两个生物学重复，首个实验（DS27）包含技术PCR重复，第二个实验（DS34）包含Drop-seq上机重复。总计生成并分析了7个文库。*** 由于涉及人类序列数据的机构限制，原始数据无法提供 ***