RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.

中文翻译：

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使用激光捕获显微切割和 Smart-3SEQ 对档案组织中的单细胞进行基因表达谱分析。

RNA 测序 (RNA-seq) 是一种灵敏且准确的基因表达定量方法。小样本或 RNA 被降解的样本，例如福尔马林固定石蜡包埋 (FFPE) 组织，使用非专门的 RNA-seq 方案进行研究仍然具有挑战性。在这里，我们提出了一种新方法 Smart-3SEQ，即使使用少量总 RNA，也能准确量化转录本丰度，并有效表征通过激光捕获显微切割 (LCM) 从 FFPE 组织中提取的小样本。我们还从 FFPE 单细胞中获得了独特的生物学特征，这是不可能用以前的 RNA-seq 方案进行研究的，并且我们使用这些数据来识别与肿瘤微环境相关的可能的新巨噬细胞表型。我们建议 Smart-3SEQ 作为一种极具成本效益的方法，使大型基因表达谱实验不受样本大小和组织可用性的限制。特别是，Smart-3SEQ 与 FFPE 组织的兼容性可释放大量存档的临床样本；与 LCM 相结合，它可以对小细胞群和通过原位环境分离的单细胞进行前所未有的研究。