The same peptide-MHC complex, however, can select multiple T cell clonotypes26. recovered CDR3 sequences, and their frequencies, of alpha and beta chains from E7-immunized mice and peanut allergy patients are available as Supplementary Tables 6, 10. Gene expression matrices for E7-immunized mice and peanut allergy patients are available as Supplementary Data 1, 2. Abstract High-throughput 3 single-cell RNA-Sequencing (scRNA-seq) allows for cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3 barcoded scRNA-seq samples. This approach is compatible with common 3 scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from food allergy patients. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) says associated with food allergy. These results demonstrate the power of our method when studying diseases in which clonotype-driven responses are crucial to understanding the underlying biology. Antigen-specific T cells play key functions CHF5074 in a number of diseases including autoimmune disorders and cancer1C3. Assessing the phenotypes and functions of these STMN1 cells is essential to both understanding underlying disease biology and designing new therapeutic modalities4,5. To study antigen-specific T cells comprehensively, two sequencing-based approaches have emerged: bulk genomic sequencing of T cell antigen receptor (repertoire thus can spotlight clonotypic diversity and the dynamics of antigen-dependent responses associated with disease, such as clonal growth or selection2,6,7. RNA-seq, in contrast, can reveal novel says and functions of disease-relevant T cells CHF5074 through unique patterns of gene expression, albeit without determination of whether those cells are recognizing common antigens8C10. Coupling these two types of data is usually of great interest for modeling the dynamics of T cell responses and isolating those cells most relevant to disease says11C13. Currently, the preferred method for linking these steps relies on sorting single T cells into multi-well plates by flow cytometry, performing full-length scRNA-seq, and then reconstructing the sequences of rearranged and genes. This strategy is limited in throughput (~10C1,000 cells) by cost, labor and time6,14,15. Recently developed high-throughput scRNA-seq methods can profile the transcriptomes of 103C105 single CHF5074 cells at once, but accomplish this task by first barcoding mRNAs on their 3 ends during reverse transcription followed by quantification of gene expression by sequencing only those 3 ends16C18. While sufficient to enumerate mRNA abundances, this process hinders precise, direct sequencing of recombined genes because the variable regions of those transcriptsparticularly the complementarity-determining region 3 (transcripts to directly enrich CDR3 sequences eliminate reverse-transcription-appended cellular barcodes and unique molecular identifiers (UMIs) positioned on the 3 ends of transcripts during amplification, and thus obscure the single-cell resolution of the data. New approaches have emerged to determine clonotypes from high-throughput 3 or 5 scRNA-seq libraries. These typically rely on specialized RNA-capture reagents (e.g., the customized transcript capture beads of DART-seq or specific kits for InDrop, Dolomite and 10X), limiting their adoption and application to previously CHF5074 archived samples. Some also require combinations of different sequencing technologies (e.g., Illumina and Nanopore in RAGE-seq), complicating their implementation11,19C23. Methods that allow for cost-efficient and simple recovery of sequences from 3 scRNA-seq libraries would enable the study of clonotypic T cell responses with confidence. RESULTS sequences recovered via targeted sequencing Here, we report a simple process to sequence concomitantly both the transcriptome and and sequences of T cells from a single sequencing library generated using a massively-parallel 3 scRNA-seq platform, such as Seq-Well or Drop-seq (Fig. 1). Our approach both overcomes the 3 bias CHF5074 and maintains the single-cell resolution in the sequencing library introduced by these platforms (Supplementary Fig. 1a,b). In our approach, a 3 barcoded whole transcriptome amplification (WTA) is performed using standard protocols for Seq-Well or Drop-seq16,18,24. Next, one fraction of.