Enriching and Characterizing T Cell Repertoires from 3' Barcoded Single-Cell Whole Transcriptome Amplification Products

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

doi:10.1007/978-1-0716-2712-9_7

Enriching and Characterizing T Cell Repertoires from 3' Barcoded Single-Cell Whole Transcriptome Amplification Products

Methods Mol Biol. 2022:2574:159-182. doi: 10.1007/978-1-0716-2712-9_7.

Authors

Tasneem Jivanjee^#^{1

2

3

4}, Samira Ibrahim^#^{1

2

3

4}, Sarah K Nyquist^{1

2

3

4

5

6}, G James Gatter^{1

2

3

4}, Joshua D Bromley^{1

2

3

4

7}, Swati Jaiswal⁸, Bonnie Berger^{4

5}, Samuel M Behar⁸, J Christopher Love^{9

10

11

12}, Alex K Shalek^{13

14

15

16

17}

Affiliations

¹ Institute for Medical Engineering & Science and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA.
² Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
³ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁵ Program in Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁶ Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁷ Microbiology Graduate Program, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁸ Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA.
⁹ Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
¹⁰ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA. [email protected].
¹¹ Broad Institute of MIT and Harvard, Cambridge, MA, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
¹² Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
¹³ Institute for Medical Engineering & Science and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
¹⁴ Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
¹⁵ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA. [email protected].
¹⁶ Broad Institute of MIT and Harvard, Cambridge, MA, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
¹⁷ Program in Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].

^# Contributed equally.

Abstract

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.

Keywords: Gene expression; Seq-Well S3; Single-cell RNA-sequencing; Single-cell TCR sequencing; T cell receptor repertoire profiling; Targeted enrichment; scRNA-seq.

MeSH terms

Receptors, Antigen, T-Cell / genetics
Sequence Analysis, RNA / methods
Single-Cell Analysis / methods
T-Lymphocytes*
Transcriptome*

Substances

Receptors, Antigen, T-Cell

Abstract

MeSH terms

Substances

Grants and funding