Comparative Toxicotranscriptomics of Single Cell RNA-Seq and Conventional RNA-Seq in TCDD-Exposed Testicular Tissue.
Comparative Toxicotranscriptomics of Single Cell RNA-Seq and Conventional RNA-Seq in TCDD-Exposed Testicular Tissue.
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DOI：
10.3389/ftox.2022.821116
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发表时间：
2022
影响因子：
--
通讯作者：
Baker, Tracie R.
中科院分区：
文献类型：
--
作者：
Haimbaugh, Alex;Meyer, Danielle;Akemann, Camille;Gurdziel, Katherine;Baker, Tracie R.
在这份报告中，我们比较了在性分化过程中暴露于2，3，7，8-四氯二苯并-对二恶英(TCDD)的成年斑马鱼睾丸的两种转录转录研究方法的结果和局限性：常规或批量RNA-seq(Bulk-seq)和单细胞RNA测序(scRNA-seq)数据。ScRNA-seq已经成为揭示存在于异质组织中的细胞类型特异性转录组动力学的有价值的工具。我们的实验室以前展示了scRNA-seq管道的毒理学价值，以表征TCDD暴露在睾丸中的后遗症，表明精子细胞和精子的丢失，而不是其他细胞类型的丢失，导致成年雄性斑马鱼在性分化期间暴露在不育的病理中。为了研究scRNA-seq中可能存在的技术缺陷，如细胞分离效应和转录组覆盖率降低，我们比较了来自对照和TCDD暴露样本的批量测序和scRNA序列配对样本，以了解scRNA-seq与本体-seq在转录和毒理学上的得失。我们推测，睾丸可能对组织破坏敏感，因为它们包含持续分裂和/或成熟的多种细胞类型，TCDD暴露可能调节了敏感性的程度。因此，我们试图了解这种解离对scRNA-seq返回的数据的毒理学价值的影响程度。我们确认，单个细胞从完整组织中所需的分离对基因表达有重大影响，影响基因路径，如果发现没有得到很好的控制和背景，可能会混淆关于暴露的毒素基因组学研究。此外，一种常见的scRNA-seq方法使用从mRNA3‘端扩增的cDNA来检测包括转录因子在内的低表达转录物。我们证实了这一点，并表明TCDD相关基因可能被scRNA-seq忽略，然而，这种欠检测效应不是由TCDD暴露介导的。即便如此，在本研究中，scRNA-seq通常比Bulk-seq方法更好地提取毒理学相关信息。这份报告旨在通过展示从测序细胞中提取的差异信息-尽管来自相同的组织和暴露方案-受到所使用的特定方案的影响，从而为未来在不断增长的毒物基因组学领域进行转录研究的实验设计提供信息，并对暴露诱导的风险进行解释。
In this report, we compare the outcomes and limitations of two methods of transcriptomic inquiry on adult zebrafish testes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during sexual differentiation: conventional or bulk RNA-seq (bulk-seq) and single cell RNA sequencing (scRNA-seq) data. scRNA-seq has emerged as a valuable tool for uncovering cell type-specific transcriptome dynamics which exist in heterogeneous tissue. Our lab previously showed the toxicological value of the scRNA-seq pipeline to characterize the sequelae of TCDD exposure in testes, demonstrating that loss of spermatids and spermatozoa, but not other cell types, contributed to the pathology of infertility in adult male zebrafish exposed during sexual differentiation. To investigate the potential for technical artifacts in scRNA-seq such as cell dissociation effects and reduced transcriptome coverage, we compared bulk-sequenced and scRNA-seq-paired samples from control and TCDD-exposed samples to understand what is gained and lost in scRNA-seq vs bulk-seq, both transcriptomically and toxicologically. We hypothesized that the testes may be sensitive to tissue disruption as they contain multiple cell types under constant division and/or maturation, and that TCDD exposure may mediate the extent of sensitivity. Thus, we sought to understand the extent to which this dissociation impacts the toxicological value of data returned from scRNA-seq. We confirm that the required dissociation of individual cells from intact tissue has a significant impact on gene expression, affecting gene pathways with the potential to confound toxicogenomics studies on exposures if findings are not well-controlled and well-situated in context. Additionally, a common scRNA-seq method using cDNA amplified from the 3’ end of mRNA under-detects low-expressing transcripts including transcription factors. We confirm this, and show TCDD-related genes may be overlooked by scRNA-seq, however, this under-detection effect is not mediated by TCDD exposure. Even so, scRNA-seq generally extracted toxicologically relevant information better than the bulk-seq method in the present study. This report aims to inform future experimental design for transcriptomic investigation in the growing field of toxicogenomics by demonstrating the differential information extracted from sequencing cells—despite being from the same tissue and exposure scheme—is influenced by the specific protocol used, with implications for the interpretation of exposure-induced risk.
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Seveso事故：查看40年的健康研究及其他地区。
影响因子：
11.8
作者：
Eskenazi B;Warner M;Brambilla P;Signorini S;Ames J;Mocarelli P
通讯作者：
Mocarelli P
影响因子：
44.1
作者：
Guo J;Grow EJ;Mlcochova H;Maher GJ;Lindskog C;Nie X;Guo Y;Takei Y;Yun J;Cai L;Kim R;Carrell DT;Goriely A;Hotaling JM;Cairns BR
通讯作者：
Cairns BR
影响因子：
11.8
作者：
Green CD;Ma Q;Manske GL;Shami AN;Zheng X;Marini S;Moritz L;Sultan C;Gurczynski SJ;Moore BB;Tallquist MD;Li JZ;Hammoud SS
通讯作者：
Hammoud SS
影响因子：
82.9
作者：
Deng Q;Han G;Puebla-Osorio N;Ma MCJ;Strati P;Chasen B;Dai E;Dang M;Jain N;Yang H;Wang Y;Zhang S;Wang R;Chen R;Showell J;Ghosh S;Patchva S;Zhang Q;Sun R;Hagemeister F;Fayad L;Samaniego F;Lee HC;Nastoupil LJ;Fowler N;Eric Davis R;Westin J;Neelapu SS;Wang L;Green MR
通讯作者：
Green MR
影响因子：
9.5
作者：
Davies P;Jones M;Liu J;Hebenstreit D
通讯作者：
Hebenstreit D