Histology-associated transcriptomic heterogeneity in ovarian folliculogenesis revealed by quantitative single-cell RNA-sequencing for tissue sections with DRaqL
Histology-associated transcriptomic heterogeneity in ovarian folliculogenesis revealed by quantitative single-cell RNA-sequencing for tissue sections with DRaqL
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DOI：
10.1101/2022.12.14.520513
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发表时间：
2022-12
期刊：
影响因子：
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通讯作者：
Hiroki Ikeda;Shintaro Miyao;So I. Nagaoka;Takuya Yamamoto;K. Kurimoto
中科院分区：
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作者：
Hiroki Ikeda;Shintaro Miyao;So I. Nagaoka;Takuya Yamamoto;K. Kurimoto
具有空间分辨率的高质量单细胞 RNA 测序 (RNA-seq) 仍然具有挑战性。激光捕获显微切割 (LCM) 是一种广泛使用的有效方法，可从组织切片中分离任意目标细胞以进行全面的转录组学。在这里，我们开发了 DRaqL（LCM 的直接 RNA 回收和猝灭），这是一种实验方法，用于有效裂解通过 LCM 从酒精和福尔马林固定切片中分离的单细胞，无需 RNA 纯化。单细胞 RNA-seq 与 DRaqL 相结合，可以对酒精固定的切片进行转录组分析，其效率与对新鲜分离的细胞进行分析的效率相当，同时还可以进行有效的外显子-外显子连接分析。此外，DRaqL 和蛋白酶处理的结合使得能够对用福尔马林牢固固定的组织切片进行稳健且高效的单细胞转录组分析。将该方法应用于小鼠卵巢切片，我们揭示了与卵母细胞大小定量相关的生长卵母细胞的转录组连续体，并检测了卵母细胞特异性剪接亚型。此外，我们的统计模型揭示了卵母细胞转录组与其大小之间关系的异质性，从而识别出卵母细胞子集中的大小与转录组关系异常。最后，我们鉴定了颗粒细胞中差异表达的基因，这些基因与颗粒细胞与卵母细胞的组织学关系相关，这表明控制生殖-体关系的不同表观遗传调控和细胞周期活动。因此，我们开发了一种多功能、有效的方法，用于从组织切片中进行稳健的单细胞 cDNA 扩增，并提供了有利于高质量转录组学的实验平台，从而揭示了卵巢组织卵泡发生中与组织学相关的转录组异质性。
High-quality single-cell RNA-sequencing (RNA-seq) with spatial resolution remains challenging. Laser capture microdissection (LCM) is a widely used, potent approach to isolate arbitrarily targeted cells from tissue sections for comprehensive transcriptomics. Here, we developed DRaqL (direct RNA recovery and quenching for LCM), an experimental approach for efficient lysis of single cells isolated by LCM from alcohol- and formalin-fixed sections without RNA purification. Single-cell RNA-seq combined with DRaqL allowed transcriptomic profiling from alcohol-fixed sections with efficiency comparable to that of profiling from freshly dissociated cells, together with effective exon– exon junction profiling. Furthermore, the combination of DRaqL and protease treatment enabled robust and efficient single-cell transcriptome analysis from tissue sections strongly fixed with formalin. Applying this method to mouse ovarian sections, we revealed a transcriptomic continuum of growing oocytes quantitatively associated with oocyte size, and detected oocyte-specific splice isoforms. In addition, our statistical model revealed heterogeneity of the relationship between the transcriptome of oocytes and their size, resulting in identification of a size–transcriptome relationship anomaly in a subset of oocytes. Finally, we identified genes that were differentially expressed in granulosa cells in association with the histological affiliations of granulosa cells to the oocytes, suggesting distinct epigenetic regulations and cell-cycle activities governing the germ–soma relationship. Thus, we developed a versatile, efficient approach for robust single-cell cDNA amplification from tissue sections and provided an experimental platform conducive to high-quality transcriptomics, thereby revealing histology-associated transcriptomic heterogeneity in folliculogenesis in ovarian tissues.