Sample Multiplexing Research Articles

A cost-effective protocol for single-cell RNA sequencing of human skin.

Single-cell RNA sequencing (scRNAseq) and flow cytometry studies in skin are methodologically complex and costly, limiting their accessibility to researchers worldwide. Ideally, RNA and protein-based analyses should be performed on the same lesion to obtain more comprehensive data. However, current protocols generally focus on either scRNAseq or flow cytometry of healthy skin. We present a novel label-free sample multiplexing strategy, building on the souporcell algorithm, which enables scRNAseq analysis of paired blood and skin samples. Additionally, we provide detailed instructions for simultaneous flow cytometry analysis from the same sample, with necessary adaptations for both healthy and inflamed skin specimens. This tissue multiplexing strategy mitigates technical batch effects and reduces costs by 2-4 times compared to existing protocols. We also demonstrate the effects of varying enzymatic incubation durations (1, 3, and 16 hours, with and without enzyme P) on flow cytometry outcomes. Comprehensive explanations of bioinformatic demultiplexing steps and a detailed step-by-step protocol of the entire experimental procedure are included. The protocol outlined in this article will make scRNAseq and flow cytometry analysis of skin samples more accessible to researchers, especially those new to these techniques.

Sample multiplexing for retinal single-cell RNA sequencing

Sample multiplexing for retinal single-cell RNA sequencing

Sample multiplexing in CyTOF: Path to optimize single-cell proteomic profiling

Sample multiplexing in CyTOF: Path to optimize single-cell proteomic profiling

Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues.

Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer's disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.

On the utility of ultrafast MS1-only proteomics in drug target discovery studies based on thermal proteome profiling method.

Advances in high-throughput high-resolution mass spectrometry and the development of thermal proteome profiling approach (TPP) have made it possible to accelerate a drug target search. Since its introduction in 2014, TPP quickly became a method of choice in chemical proteomics for identifying drug-to-protein interactions on a proteome-wide scale and mapping the pathways of these interactions, thus further elucidating the unknown mechanisms of action of a drug under study. However, the current TPP implementations based on tandem mass spectrometry (MS/MS), associated with employing lengthy peptide separation protocols and expensive labeling techniques for sample multiplexing, limit the scaling of this approach for the ever growing variety of drug-to-proteomes. A variety of ultrafast proteomics methods have been developed in the last couple of years. Among them, DirectMS1 provides MS/MS-free quantitative proteome-wide analysis in 5-min time scale, thus opening the way for sample-hungry applications, such as TPP. In this work, we demonstrate the first implementation of the TPP approach using the ultrafast proteome-wide analysis based on DirectMS1. Using a drug topotecan, which is a known topoisomerase I (TOP1) inhibitor, the feasibility of the method for identifying drug targets at the whole proteome level was demonstrated for an ovarian cancer cell line.

Evaluation of a method to fluorescently label platelets for in-human recovery and survival studies.

Platelets for transfusion are evaluated for in vivo quality using recovery and survival measurements in healthy human subjects. Radiolabelling is the standard for tracing platelets post-transfusion but imposes logistical and technical limitations. This study investigates the in vitro feasibility of labelling platelets with the calcein family of fluorescent dyes as an alternative to radioisotopes or biotin. Protocols for radiolabelling were adapted for use with calcein acetoxymethyl ester (CAM) and biotin. Labelled platelets were analysed by flow cytometry and evaluated for activation and function. We tested feasibility for labelling without manipulation of platelets and for multiplexing of samples. Labelling at 2 μg CAM/1010 platelets resulted in >99% of CAM+ platelets. There was no significant difference in activation or aggregation between CAM-labelled or biotinylated platelets and vehicle controls although %CD62P+ was significantly lower in platelets that were not processed for labelling. Addition of CAM to the platelet storage bag labelled >95% of platelets. Platelet populations labelled with different dyes could be distinguished by flow cytometry. These data provide a rationale for further development of CAM and other fluorescent dyes as tools for measuring post-transfusion kinetics of platelets.

Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample Multiplexing.

GoDig, a platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standards, is a powerful, flexible, and easy-to-use method that uses tandem mass tags to increase sample throughput up to 18-fold relative to label-free methods. Though the protein-level success rates of GoDig are high, the peptide-level success rates are more limited, hampering assays of harder-to-quantify proteins and site-specific phenomena. To guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of "priming runs," an acquisition mode with significantly higher success rates. In this mode, two or more chromatographic priming runs are automatically performed to improve the accuracy and precision of target elution orders, followed by analytical runs which quantify targets. Using priming runs, success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy expression profiles across four human cell lines.

The Transpeptidase Sortase A Binds Nucleic Acids and Mediates Mammalian Cell Labeling.

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.

Comparative Analysis of Single-Cell RNA Sequencing Methods with and without Sample Multiplexing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful technique for investigating biological heterogeneity at the single-cell level in human systems and model organisms. Recent advances in scRNA-seq have enabled the pooling of cells from multiple samples into single libraries, thereby increasing sample throughput while reducing technical batch effects, library preparation time, and the overall cost. However, a comparative analysis of scRNA-seq methods with and without sample multiplexing is lacking. In this study, we benchmarked methods from two representative platforms: Parse Biosciences (Parse; with sample multiplexing) and 10x Genomics (10x; without sample multiplexing). By using peripheral blood mononuclear cells (PBMCs) obtained from two healthy individuals, we demonstrate that demultiplexed scRNA-seq data obtained from Parse showed similar cell type frequencies compared to 10x data where samples were not multiplexed. Despite relatively lower cell capture affecting library preparation, Parse can detect rare cell types (e.g., plasmablasts and dendritic cells) which is likely due to its relatively higher sensitivity in gene detection. Moreover, a comparative analysis of transcript quantification between the two platforms revealed platform-specific distributions of gene length and GC content. These results offer guidance for researchers in designing high-throughput scRNA-seq studies.

Abstract 333: Stability and performance of a partially used 8-lane microwell cartridge on single-cell capture and immune response profiling

Abstract In this study, we introduce an innovative microwell-based approach, offering the capability to capture up to 500,000 single cells and show stability of a partially used 8-lane microwell cartridge up to 6 months after first use. Stability of microwell cartridges used for single-cell capture and the integrity of the individual lanes are crucial factors in genomics research to ensure the accuracy and reliability of generated data. Stability is paramount to ensure consistent results over multiple experiments. High-quality materials and manufacturing processes are essential to prevent wear and tear, ensuring that the microwell cartridges function optimally for an extended period. Integrity between the individual lanes must also be intact to prevent cross-contamination between samples. To demonstrate partial use stability of the 8-lane microwell cartridge, multiple assay combinations were performed on different lanes of the cartridge, collected at different time points. Whole transcriptome amplification (WTA) together with AbSeq antibody-oligonucleotide conjugates for cellular indexing of transcriptomes and epitopes (CITE-Seq) was performed on peripheral blood mononuclear cells (PBMCs), loaded and captured in Lanes 1 and 2, on Day 1; sample multiplexing was used with targeted mRNA assay on Jurkat and Ramos cells, loaded and captured in Lanes 3 and 4, after four months; and WTA together with the AbSeq Assay was performed on PBMCs, loaded and captured in Lanes 5 and 6, after six months. The different time points collected were compared to a single-lane microwell cartridge. Our data showed that assay performance was not compromised with the storage of a partially used cartridge, and there was no contamination detected between the lanes. High-throughput cell load and capture using WTA and the AbSeq Assay also showed that each lane of the 8-lane microwell cartridge was able to process up to 65,000 cells with no limit of detection on assay sensitivity. By maintaining cartridge stability and stringent measures to prevent contamination, researchers can ensure the reliability and accuracy of their data. This is vital for various applications such as developing targeted immunotherapies and precision medicine approaches, ultimately enhancing our capacity to combat a wide range of diseases. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. Citation Format: Hye-Won Song, Ricelle A. Acob, Zorine Hlathu, Xueying Zhao, Jamie Moskwa, Larry Wang, Aruna Ayer. Stability and performance of a partially used 8-lane microwell cartridge on single-cell capture and immune response profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 333.

Abstract 6333: A high-parameter mass cytometry panel for the functional characterization of CAR T cells

Abstract High-parameter flow cytometry provides a powerful tool for in-depth analysis of CAR T cell therapy, from characterizing CAR T cell populations to understanding their interactions within the tumor microenvironment. This information is crucial for advancing CAR T cell therapies and improving their effectiveness in treating cancer. To address the need for deep immunoprofiling at all stages of CAR T cell therapy, we created a robust and comprehensive mass cytometry panel of antibodies against 44 cell surface and intracellular markers. This assay allows for comprehensive characterization of CAR T cell features including surface marker expression, activation state, cytokine production, and differentiation status. These data will provide researchers with a better understanding of the phenotype of the CAR T cells used in therapy and can guide optimization efforts to enhance expansion, persistence, and self-renewal. For example, newer-generation CAR T cells (T cells redirected for antigen-unrestricted cytokine-initiated killing, or TRUCKs) that are engineered to produce cytokines such as IL-2 to enhance their own survival can be evaluated in vitro to select CAR constructs with the most potent anti-tumor activity. The lyophilized and validated 30-marker Maxpar® Direct™ Immune Profiling Assay™ enables comprehensive characterization of immune cell populations in both whole blood and PBMC. The 7-marker Maxpar Direct T Cell Expansion Panel 3 (OX40, TIGIT, CD69, PD-1, Tim-3, ICOS, and 4-1BB) and the Maxpar Direct Basic Activation Expansion Panel (CD107a, IL-2, TNFα, IFNγ, perforin, and granzyme B) are add-on modules that can further characterize CAR T cell exhaustion, degranulation, cytokine production, and cytotoxicity against target cells. We also compared CAR T detection options for sensitivity and specificity using a CD19 CAR-transduced cell line spiked into healthy donor PBMC. Indirect staining with Miltenyi Biotec biotinylated CD19 CAR Detection Reagent followed by anti-biotin- or streptavidin-conjugated metal tags was compared against a directly conjugated or biotinylated anti-G4S linker antibody. We included sample multiplexing using 6 metal-tagged CD45 antibodies to address the need to minimize batch effects, crucial to multi-site and longitudinal studies. Samples were acquired on a CyTOF® XT™ instrument, and preliminary data analysis was performed using Maxpar Pathsetter™ for automated enumeration of immune cells. By combining the lyophilized single-tube Maxpar Immune Profiling Assay and Expansion Panels with specific detection of CAR T cells, this assay enables researchers to analyze multiple key parameters simultaneously at the single-cell level at all stages of CAR T therapy development. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Deeqa Mahamed, Geneve Awong, Thiru Selvanatham. A high-parameter mass cytometry panel for the functional characterization of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6333.

DeMULTIplex2: robust sample demultiplexing for scRNA-seq

Sample multiplexing enables pooled analysis during single-cell RNA sequencing workflows, thereby increasing throughput and reducing batch effects. A challenge for all multiplexing techniques is to link sample-specific barcodes with cell-specific barcodes, then demultiplex sample identity post-sequencing. However, existing demultiplexing tools fail under many real-world conditions where barcode cross-contamination is an issue. We therefore developed deMULTIplex2, an algorithm inspired by a mechanistic model of barcode cross-contamination. deMULTIplex2 employs generalized linear models and expectation–maximization to probabilistically determine the sample identity of each cell. Benchmarking reveals superior performance across various experimental conditions, particularly on large or noisy datasets with unbalanced sample compositions.

Biomimetic Synthesis and Chemical Proteomics Reveal the Mechanism of Action and Functional Targets of Phloroglucinol Meroterpenoids.

Natural products perennially serve as prolific sources of drug leads and chemical probes, fueling the development of numerous therapeutics. Despite their scarcity, natural products that modulate protein function through covalent interactions with lysine residues hold immense potential to unlock new therapeutic interventions and advance our understanding of the biological processes governed by these modifications. Phloroglucinol meroterpenoids constitute one of the most expansive classes of natural products, displaying a plethora of biological activities. However, their mechanism of action and cellular targets have, until now, remained elusive. In this study, we detail the concise biomimetic synthesis, computational mechanistic insights, physicochemical attributes, kinetic parameters, molecular mechanism of action, and functional cellular targets of several phloroglucinol meroterpenoids. We harness synthetic clickable analogues of natural products to probe their disparate proteome-wide reactivity and subcellular localization through in-gel fluorescence scanning and cell imaging. By implementing sample multiplexing and a redesigned lysine-targeting probe, we streamline a quantitative activity-based protein profiling, enabling the direct mapping of global reactivity and ligandability of proteinaceous lysines in human cells. Leveraging this framework, we identify numerous lysine-meroterpenoid interactions in breast cancer cells at tractable protein sites across diverse structural and functional classes, including those historically deemed undruggable. We validate that phloroglucinol meroterpenoids perturb biochemical functions through stereoselective and site-specific modification of lysines in proteins vital for breast cancer metabolism, including lipid signaling, mitochondrial respiration, and glycolysis. These findings underscore the broad potential of phloroglucinol meroterpenoids for targeting functional lysines in the human proteome.

Expression of pro- and anti-inflammatory cytokines during anti-proprotein convertase subtilisin/kexin type 9 therapy in patients with statin-resistant familial hypercholesterolemia.

Some clinical dyslipidemia cases do not respond to statins, known as statin-resistant familial hypercholesterolemia (SR-FH), in which patients are under a high cardiovascular risk despite statin therapy. Therefore, novel therapeutic alternatives are required. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) reduce cholesterol levels and cardiovascular disease risk, particularly in patients with SR-FH, where PCSK9i may differentially affect pro- and anti-inflammatory mediators depending on the clinical setting. To evaluate the effect of PCSK9i treatment on pro- and anti-inflammatory cytokines in patients with SR-FH. Before-after comparison, quasi-experimental, single-center study in patients with SR-FH. Blood samples were processed to obtain complete blood counts of glycated hemoglobin and serum lipid levels. Flow cytometry was performed to characterize baseline circulating M1- and M2-macrophages and monocytes. Multiplexing of plasma samples was used to compare plasma fraktaline, interleukins (ILs), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-alpha. The endpoints were lower serum lipid levels and pro-inflammatory mediator modification. Twenty patients with SR-FH, aged 58 years and most of them males, were included, with a mean body-mass index of 26.4 and showing ischemic heart disease and similar values of baseline M1- and M2-macrophages and monocytes. Six-month iPSCK-9 therapy considerably reduced LDLc, increased anti-inflammatory cytokine (IL-4), and modified pro-inflammatory cytokine (TNF-alpha and MCP-1) levels. No notable effects were observed for the other markers. PCSK9i therapy exerted subclinical anti-inflammatory and anti-atherogenic effects, indicating potential benefits for clinical outcomes.

A comprehensive assessment of exome capture methods for RNA sequencing of formalin-fixed and paraffin-embedded samples

RNA-Seq analysis of Formalin-Fixed and Paraffin-Embedded (FFPE) samples has emerged as a highly effective approach and is increasingly being used in clinical research and drug development. However, the processing and storage of FFPE samples are known to cause extensive degradation of RNAs, which limits the discovery of gene expression or gene fusion-based biomarkers using RNA sequencing, particularly methods reliant on Poly(A) enrichment. Recently, researchers have developed an exome targeted RNA-Seq methodology that utilizes biotinylated oligonucleotide probes to enrich RNA transcripts of interest, which could overcome these limitations. Nevertheless, the standardization of this experimental framework, including probe designs, sample multiplexing, sequencing read length, and bioinformatic pipelines, remains an essential requirement. In this study, we conducted a comprehensive comparison of three main commercially available exome capture kits and evaluated key experimental parameters, to provide the overview of the advantages and limitations associated with the selection of library preparation protocols and sequencing platforms. The results provide valuable insights into the best practices for obtaining high-quality data from FFPE samples.

High-capacity sample multiplexing for single cell chromatin accessibility profiling

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

Proteome-Wide Profiling Using Sample Multiplexing of a Human Cell Line Treated with Cannabidiol (CBD) and Tetrahydrocannabinol (THC).

Cannabis has been used historically for both medicinal and recreational purposes, with the most notable cannabinoids being cannabidiol (CBD) and tetrahydrocannabinol (THC). Although their therapeutic effects have been well studied and their recreational use is highly debated, the underlying mechanisms of their biological effects remain poorly defined. In this study, we use isobaric tag-based sample multiplexed proteome profiling to investigate protein abundance differences in the human neuroblastoma SH-SY5Y cell line treated with CBD and THC. We identified significantly regulated proteins by each treatment and performed a pathway classification and associated protein-protein interaction analysis. Our findings suggest that these treatments may lead to mitochondrial dysfunction and induce endoplasmic reticulum stress. These data can potentially be interrogated further to investigate the potential role of CBD and THC in various biological and disease contexts, providing a foundation for future studies.

Accessible high-throughput single-cell whole-genome sequencing with paired chromatin accessibility

Single-cell whole-genome sequencing (scWGS) enables the assessment of genome-level molecular differences between individual cells with particular relevance to genetically diverse systems like solid tumors. The application of scWGS was limited due to a dearth of accessible platforms capable of producing high-throughput profiles. We present a technique that leverages nucleosome disruption methodologies with thewidely adopted 10× Genomics ATAC-seq workflow to produce scWGS profiles for high-throughput copy-number analysis without new equipment or custom reagents. We further demonstrate the use of commercially available indexed transposase complexes from ScaleBio for sample multiplexing, reducing the per-sample preparation costs. Finally, we demonstrate that sequential indexed tagmentation with an intervening nucleosome disruption step allows for the generation of both ATAC and WGS data from the same cell, producing comparable data to the unimodal assays. By exclusively utilizing accessible commercial reagents, we anticipate that these scWGS and scWGS+ATAC methods can be broadly adopted by the research community.

Proteome-wide abundance profiling of yeast deletion strains for GET pathway members using sample multiplexing.

The GET pathway is associated with post-translational delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) in yeast, as well as other eukaryotes. Moreover, dysfunction of the GET pathway has been associated with various pathological conditions (i.e., neurodegenerative disorders, cardiovascular ailments, and protein misfolding diseases). In this study, we used yeast deletion strains of Get complex members (specifically, Get1, Get2, Get3, Get4, and Get5) coupled with sample multiplexing-based quantitative mass spectrometry to profile protein abundance on a proteome-wide scale across the five individual deletion strains. Our dataset consists of over 4500 proteins, which corresponds to >75% of the yeast proteome. The data reveal several dozen proteins that are differentially abundant in one or more deletion strains, some of which are membrane-associated, yet the abundance of many TA proteins remained unchanged. This study provides valuable insights into the roles of these Get genes, and the potential for alternative pathways which help maintain cellular function despite the disruption of the GET pathway.

Using combined single-cell gene expression, TCR sequencing and cell surface protein barcoding to characterize and track CD4+ T cell clones from murine tissues.

Single-cell gene expression analysis using sequencing (scRNA-seq) has gained increased attention in the past decades for studying cellular transcriptional programs and their heterogeneity in an unbiased manner, and novel protocols allow the simultaneous measurement of gene expression, T-cell receptor clonality and cell surface protein expression. In this article, we describe the methods to isolate scRNA/TCR-seq-compatible CD4+ T cells from murine tissues, such as skin, spleen, and lymph nodes. We describe the processing of cells and quality control parameters during library preparation, protocols for multiplexing of samples, and strategies for sequencing. Moreover, we describe a step-by-step bioinformatic analysis pipeline from sequencing data generated using these protocols. This includes quality control, preprocessing of sequencing data and demultiplexing of individual samples. We perform quantification of gene expression and extraction of T-cell receptor alpha and beta chain sequences, followed by quality control and doublet detection, and methods for harmonization and integration of datasets. Next, we describe the identification of highly variable genes and dimensionality reduction, clustering and pseudotemporal ordering of data, and we demonstrate how to visualize the results with interactive and reproducible dashboards. We will combine different analytic R-based frameworks such as Bioconductor and Seurat, illustrating how these can be interoperable to optimally analyze scRNA/TCR-seq data of CD4+ T cells from murine tissues.