Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
Nat Protoc. 2016 Mar;11(3):429-42. doi: 10.1038/nprot.2016.024. Epub 2016 Feb 4.
High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >10(4) lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample.
高通量测序免疫受体的可变区(抗体和 T 细胞受体(TCR))在理解健康和疾病中的适应性免疫反应方面具有重要意义。然而,对于包含>10(4)个淋巴细胞的典型样本,在单细胞水平上同时对两条免疫受体链(VH+VL 或 TCRβ/δ+TCRα/γ)进行测序是有问题的,因为免疫受体由两条分别由独立 mRNA 编码的多肽链组成。本文介绍了一种技术,可从数百万个细胞中以高精度(>97%)快速且低成本地确定配对免疫受体库。流聚焦用于将单个细胞包裹在含有磁珠的乳液中,以捕获 mRNA。然后将 mRNA 转录本反转录,通过重叠延伸 PCR 将其与其伙伴物理连接,并通过高通量配对末端 Illumina 测序进行检测。该方案详细描述了流聚焦装置的构建和操作,以及用于解释数据的生物信息学管道。整个过程可以由单个研究人员在每个样本 12 小时内完成。