T细胞受体
生物
计算生物学
单细胞测序
底漆(化妆品)
转录组
大规模并行测序
DNA测序
T细胞
遗传学
基因
表型
外显子组测序
有机化学
化学
基因表达
免疫系统
作者
Tasneem Jivanjee,Samira Ibrahim,Sarah K. Nyquist,G. James Gatter,Joshua D. Bromley,Swati Jaiswal,Bonnie Berger,Samuel M. Behar,J. Christopher Love,Alex K. Shalek
出处
期刊:Methods in molecular biology
日期:2022-01-01
卷期号:: 159-182
被引量:2
标识
DOI:10.1007/978-1-0716-2712-9_7
摘要
Antigen-specific T cells play an essential role in immunoregulation and many diseases such as cancer. Characterizing the T cell receptor (TCR) sequences that encode T cell specificity is critical for elucidating the antigenic determinants of immunological diseases and designing therapeutic remedies. However, methods of obtaining single-cell TCR sequencing data are labor and cost intensive, typically requiring both cell sorting and full-length single-cell RNA-sequencing (scRNA-seq). New high-throughput 3' cell-barcoding scRNA-seq methods can simplify and scale this process; however, they do not routinely capture TCR sequences during library preparation and sequencing. While 5' cell-barcoding scRNA-seq methods can be used to examine TCR repertoire at single-cell resolution, doing so requires specialized reagents which cannot be applied to samples previously processed using 3' cell-barcoding methods.Here, we outline a method for sequencing TCRα and TCRβ transcripts from samples already processed using 3' cell-barcoding scRNA-seq platforms, ensuring TCR recovery at a single-cell resolution. In short, a fraction of the 3' barcoded whole transcriptome amplification (WTA) product typically used to generate a massively parallel 3' scRNA-seq library is enriched for TCR transcripts using biotinylated probes and further amplified using the same universal primer sequence from WTA. Primer extension using TCR V-region primers and targeted PCR amplification using a second universal primer result in a 3' barcoded single-cell CDR3-enriched library that can be sequenced with custom sequencing primers. Coupled with 3' scRNA-seq of the same WTA, this method enables simultaneous analysis of single-cell transcriptomes and TCR sequences which can help interpret inherent heterogeneity among antigen-specific T cells and salient disease biology. The method presented here can also be adapted readily to enrich and sequence other transcripts of interest from both 3' and 5' barcoded scRNA-seq WTA libraries.
科研通智能强力驱动
Strongly Powered by AbleSci AI