Highly Multiplexed Single-cell Transcript Analysis Using DNA-barcoded Nanowells

描述（由申请人提供）：单细胞转录分析已证明其识别新细胞亚群的能力，但目前受到可并行测量的细胞和分析物数量的限制。我们计划通过使用 DNA 条形码纳升孔 (nanowell) 阵列用 DNA 编码地址标记每个单独的转录本，将可监测的细胞和分析物的数量增加一到两个数量级。下一代测序将识别转录本的身份和附加的条形码，从而将每个测序的转录本追溯到单个细胞。这将通过不对称 PCR 将微阵列表面合成的 100 万个 DNA 条形码转移到纳米孔中的引物偶联纳米颗粒上来实现，同时纳米孔被微阵列表面密封。 PCR 反应还会向每个条形码添加聚 (dT) 尾。然后，单个细胞将被密封到条形码纳米孔中。细胞裂解后，聚 (dT) 探针将捕获 mRNA，逆转录酶将延伸聚 (dT) 序列，从而将条形码与每个转录物融合。条形码 cDNA 将被扩增并集成到下一代测序工作流程中。该技术将通过使用 Fluidigm 平台通过单细胞 qPCR 将测得的转录水平与同一细胞群中测得的水平进行比较来验证。我们还将通过对 B 和 T 细胞受体转录本进行测序来证明条形码可识别单个细胞转录本，并证明每个独特的 BCR 或 TCR 转录本都融合了独特的条形码，并且条形码映射回最初包含正确细胞类型的孔。此外，单细胞转录物数据将通过我们之前描述的微雕刻方法与细胞裂解前获得的相同细胞的单细胞分泌数据整合，从而建立第一个平台，可以从相同的单细胞群体中创建高度多重的单细胞转录物和蛋白质组数据。该技术的应用将大大加速我们对单细胞生物学和异质细胞群的理解。

DESCRIPTION (provided by applicant): Single cell transcriptional analysis has already demonstrated its ability to identify novel cell subsets but is currently limited by the number of cells and analytes that can be measured in parallel. We plan to increase both the number of cells and analytes that can be monitored by one to two orders of magnitude by using DNA-bar coded nanoliter well (nanowell) arrays to label each individual transcript with a DNA-encoded address. Next generation sequencing will identify both the transcript identity and the attached barcode, thereby tracing each sequenced transcript back to a single cell. This will be accomplished by transferring a million DNA barcodes synthesized on the surface of a microarray to primer-conjugated nanoparticles in the nanowells through asymmetric PCR while the nanowells are sealed by the microarray surface. The PCR reaction will also add a poly(dT) tail to each barcode. Single cells will then be sealed into the bar coded nanowells. Following lysis of the cells, the poly(dT) probe will capture the mRNA and reverse transcriptase will extend the poly(dT) sequence, thereby fusing the barcode with each transcript. The bar coded cDNA will be amplified and integrated into next generation sequencing workflows. The technique will be validated by comparing the measured transcript levels to the levels measured in the same cell population by single cell qPCR using the Fluidigm platform. We will also demonstrate that the barcodes identify individual cell transcripts by sequencing B and T cell receptor transcripts and demonstrating that each unique BCR or TCR transcript has a unique barcode fused to it and the barcode maps back to a well that originally contained the correct cell type. Furthermore, the single cell transcript data will be integrated with single cell secretion data from the same cells obtained prior to cell lysis through our previously described microengraving methodology, thereby establishing the first platform that can create highly multiplexed single cell transcript ad proteomic data from the same population of single cells. Application of this technology will greatly accelerate our understanding of single cell biology and heterogeneous cell populations.