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对来自数百万个淋巴细胞的免疫受体库进行超高通量测序。

Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes.

机构信息

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.

出版信息

Nat Protoc. 2016 Mar;11(3):429-42. doi: 10.1038/nprot.2016.024. Epub 2016 Feb 4.

Abstract

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.

摘要

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。

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