Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.
Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.
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DOI：
10.1038/s41592-022-01604-1
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发表时间：
2022-11
期刊：
影响因子：
48
通讯作者：
Yin, Peng
中科院分区：
文献类型：
--
作者：
Kishi, Jocelyn Y.;Liu, Ninning;West, Emma R.;Sheng, Kuanwei;Jordanides, Jack J.;Serrata, Matthew;Cepko, Constance L.;Saka, Sinem K.;Yin, Peng
我们提出了Light-Seq，这是一种在固定的细胞和组织中使用光定向DNA条形码，然后进行非原位测序来对完整生物样品进行多重空间索引的方法。Light-Seq将DNA条形码在互补DNA上的空间靶向快速光交联与一步DNA拼接反应相结合，以创建合并的空间索引测序文库。这种光定向条形码使得能够基于位置、形态学或蛋白质染色在完整固定的组织样品中原位选择多个细胞群体用于全转录组测序，而无需细胞解离。将Light-Seq应用于小鼠视网膜切片，我们从三个细胞层中回收了数千个差异富集的转录本，并发现了一种非常罕见的神经元亚型多巴胺能无长突细胞的生物标志物，每个切片只有4到8个细胞。Light-Seq提供了一个可访问的工作流程，将联合收割机原位成像和蛋白质染色与相同细胞的下一代测序相结合，保留完整的样品用于测序后的进一步分析。Light-Seq在固定的细胞和组织中使用光引导的DNA条形码进行多重空间索引和随后的下一代测序。这种方法融合了空间和组学信息，能够分析复杂组织中的稀有细胞类型。
We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing. Light-Seq uses light-directed DNA barcoding in fixed cells and tissues for multiplexed spatial indexing and subsequent next generation sequencing. This approach blends spatial and omics information to enable analysis of rare cell types in complex tissues.
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