The DriverMap™ Single-cell Adaptive Immune Receptor (scAIR) Immunophenotyping Assay enables identification of TCR/BCR paired full-length (V(D)J) immune receptor sequences and phenotypic characterization of single T or B cells sorted in a 96-well plate. The paired full-length receptor sequence information is necessary to clone and express functional TCRs, BCRs, and antibodies for follow-up antigen discovery studies and the development of cell-based or antibody-based therapies.
While the bulk DriverMap™ AIR RNA/DNA Repertoire Analysis Assay allows quantitative profiling of abundant clonotypes, the scAIR assays are designed to get additional full-length chain pairing information and phenotype of T- or B-cell clonotypes. This assay allows a cost-effective strategy to scRNA-seq analysis of hundreds of cells and is designed to obtain detailed immunophenotyping information for specific disease/treatment-associated subtype(s) of T- or B-cells. Hundreds of T cells of interest can be sorted in 96-well plates using a conventional FACS-based workflow, based on the expression of specific markers (e.g., activated, dysfunctional, memory, etc.), or after labeling with specific antigen or MHC-peptide complexes. This assay is available in two formats—one to identify receptors on T-cells and the other on B-cells:
- The scAIR TCR-Mark36 Assay is designed to simultaneously amplify—in a single multiplex RT-PCR reaction—full-length (CDR1-CDR2-CDR3) regions for alpha and beta (TRA and TRB) TCR chains, and 36 top T cell immunophenotyping marker mRNAs for single T cells sorted in 96-well plate(s) (see Appendix E for the list of T cell marker genes).
- The scAIR BCR-Mark30 Assay allows analysis of full-length (CDR1-CDR2-CDR3) chain pairing for all BCR genes (IGH, IGL, and IGK) and expression of 30 informative B cell immunophenotyping genes for single B cells sorted in 96-well plates (s) (see Appendix E for the list of B cell marker genes).
The amplified cDNA products for both assays contain Validator Barcodes (VBC), cell/well-specific barcodes, and P5 & P7 indexes compatible with Illumina’s Next-Generation Sequencing (NGS) to measure T-cell marker gene expression and analyze paired immune receptor chain RNA sequences at single-cell resolution.
Key Features of the DriverMap scAIR Immunophenotyping Assay:
• The reverse gene-specific primers (Rev GSPs) for both immune receptors and marker genes are designed with 10-nt cell/well-specific barcodes to allow pooling of 96 cells from each plate in a single test tube immediately after the initial Rev GSP-RNA hybridization step. This protocol design significantly simplifies the workflow, allows us to simultaneously process several plates, and reduces library preparation time for follow-up reverse transcription and PCR steps.
• The reverse GSPs are also designed with VBCs, which facilitate the accurate quantification of the copy number of cDNA molecules used in amplification steps and the selection of correct immune receptor chains from mutated sequences generated at amplification and NGS steps.
• The scAIR Immunophenotyping assay works with FACS-sorted cells in a 96-well plate without needing special microfluidic equipment or complex split-pool barcoding workflows.
• The scAIR Immunophenotyping assay employs several mechanisms to minimize off-target and primer dimer background product amplification.
• The kit contains control PBMC RNA that can be run in parallel for accurate quantitation. DriverMap Spike-in Controls can also be purchased to add to the single-cell experiment as additional controls.
• NGS data can be analyzed with open-source or MiXCR, Platforma, and RSEM software packages available online.
Related Products and Services:
- DriverMap™ AIR TCR/BCR Profiling Kit (Bulk) (human/mouse RNA/DNA)
- DriverMap™ EXP Human Genome-wide 19K Profiling Kit
- DriverMap™ Spike-in Controls
- DriverMap™ scAIR Chain Pairing Assays (without immune markers)
Visit our website for scAIR Custom Services from Cellecta.
Please read the entire user manual before proceeding with your experiment.
PLEASE NOTE: The purchase of all Cellecta Products is covered by Cellecta’s standard Terms and Conditions of Sale as described on Cellecta’s website, and selected Products containing particular technology or having certain features are also subject to restrictions on use as outlined in the Label License section of our website. Please review these Terms and Label License Restrictions before opening and using your Product and, if you are not able to abide by the restriction, contact Cellecta to return the item to Cellecta for a full refund.
Need more help with this?
Contact Us