Single-Cell Multiomics Protocols
Sample Preparation
Sample Preparation and Cell Labeling
Preparing Single Cell Suspensions (new beads)>
This protocol describes preparation of single cell suspensions of commonly used immune cell types. A single cell suspension can be loaded into a BD Rhapsody™ Cartridge. This protocol does not describe tissue dissociation.
Preparing Single Cell Suspensions (discontinued beads)
Co-staining cells with fluorescent antibodies and BD Oligo-Conjugated Antibodies>
In this technical bulletin, we address how to co-stain cells with the same clone of both fluorescent antibodies and oligonucleotide-conjugated antibodies (BD® Single-Cell Multiplexing Kit or BD® AbSeq Ab-Oligos).
Single Cell Labelling with the BD Single-Cell Multiplexing Kits>
The BD® Human Single-Cell Multiplexing Kit (Cat. No. 633781) and the BD® Mouse Immune Single-Cell Multiplexing Kit (Cat. No. 633793) utilize an innovative antibody-oligo technology to provide higher sample throughput for single cell library preparation.
Single Cell Labeling with BD® Flex Single-Cell Multiplexing Kits>
BD® Flex Single-Cell Multiplexing Kits utilize an innovative antibody-oligo technology to provide higher sample throughput for single-cell library preparation, as well as improve detection and elimination of multiplet data from single-cell experiments.
Single Cell Labelling with BD™ AbSeq Ab-Oligos (1 plex to 40 plex)>
This protocol describes use of BD AbSeq Ab-Oligos (antibody-oligonucleotides) for antigen-expression profiling with BD Rhapsody™ single cell capture and downstream library preparation.
Single-Cell Labeling with BD® AbSeq Ab-Oligos (41 plex to 100 plex)>
This protocol describes the use of BD® AbSeq Ab-Oligos (antibody-oligonucleotides) for antigen expression profiling with BD Rhapsody™ single-cell capture and downstream library preparation.
dCODE Dextramer® (RiO) staining protocol>
This protocol provides instructions on cell labeling with dCODE Dextramer® and optional BD AbSeq Ab-Oligos.
Single Cell Labelling with the BD Single-Cell Multiplexing Kit and BD® AbSeq Ab-Oligos (1 plex to 40 plex)>
This protocol describes use of BD® AbSeq Ab-Oligos (antibody-oligonucleotides) with the BD® Human Single-Cell Multiplexing Kit (Cat. No. 633781) or BD® Mouse Immune Single-Cell Multiplexing Kit (Cat. No. 633793).
Single-Cell Labeling with BD Single-Cell Multiplexing Kit and BD® AbSeq Ab-Oligos (41 plex to 100 plex)>
This protocol describes the use of BD AbSeq Ab-Oligos (antibody-oligonucleotides) (41 plex to 100 plex) with the BD® Human Single-Cell Multiplexing Kit (Cat. No. 633781).
Single-Cell Labeling with BD® Flex Single-Cell Multiplexing Kits and BD® AbSeq Ab-Oligos>
BD® AbSeq Immune Discovery Panel (IDP)>
The IDP consists of 30 different specificities against major human immune markers combined in a single tube. Designed and optimized to work on the BD Rhapsody™ Single-Cell Analysis System, the IDP is tested to work seamlessly with BD Rhapsody Single-Cell RNA Assays and Multiplexing Kits. In this brochure, we also describe steps for reconstituting the lyophilized IDP and staining cells
cDNA Synthesis Protocols
Single Cell Capture and cDNA Synthesis with the BD Rhapsody Express Single-Cell Analysis System>
This protocol describes cell loading in the BD Rhapsody™ Cartridge and single cell capture with the BD Rhapsody™ Express Single-Cell Analysis system.
Single Cell Capture and cDNA Synthesis with the BD Rhapsody Single-Cell Analysis System>
This protocol describes cell loading in the BD Rhapsody™ Cartridge and single cell capture with the BD Rhapsody™ Single-Cell Analysis system with the BD Rhapsody™ Scanner.
Processing nuclei samples on the BD Rhapsody™ Single-Cell Analysis System>
In this technical bulletin, we show how isolated nuclei can be captured and amplified on the BD Rhapsody™ Single-Cell Analysis System.
Sequencing Library Preparation Protocols
BD® AbSeq (Protein-only) Library Preparation Protocols (for AbSeq-based cell calling)
BD® AbSeq Library Preparation Protocol (for AbSeq-based cell calling)>
This protocol enables users to use cell surface proteins to profile cells independently of mRNA. Included here are instructions for how to generate BD® AbSeq single-cell libraries using the BD Rhapsody™ Single-Cell Analysis system, or the BD Rhapsody™ Express Single-Cell Analysis system. Sequencing of libraries are completed on Illumina sequencers followed by data analysis that utilizes BD® AbSeq-based cell calling pipeline which identifies cells without mRNA information.
BD® AbSeq and Sample Tag Library Preparation Protocol (for AbSeq-based cell calling)>
(WTA) Library Preparation Protocol
mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol>
This protocol provides instructions on creating a single cell whole transcriptome mRNA library after cell capture on the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina sequencers.
mRNA Whole Transcriptome Analysis (WTA) and AbSeq Library Preparation Protocol>
This protocol provides instructions on creating single cell whole transcriptome mRNA and AbSeq protein detection libraries after cell capture on the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina sequencers.
mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol>
This protocol provides instructions on creating single cell whole transcriptome mRNA and Sample Tag libraries after cell capture on the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina sequencers.
mRNA Whole Transcriptome Analysis (WTA), AbSeq, and Sample Tag Library Preparation Protocol>
This protocol provides instructions on creating single cell whole transcriptome mRNA, AbSeq, and Sample Tag libraries after cell capture on the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina sequencers.
Targeted Gene Expression Analysis Library Preparation Protocols
mRNA Targeted Library Preparation Protocol>
This protocol provides instructions on creating a single cell mRNA library after cell capture on the BD Rhapsody™ Single-Cell Analysis system or the BD Rhapsody™ Express Single-Cell Analysis system for sequencing on Illumina sequencers.
mRNA Targeted and Sample Tag Library Preparation Protocol>
This protocol provides instructions on creating single cell mRNA and Sample Tag libraries after cell capture on the BD Rhapsody™ Single-Cell Analysis system or the BD Rhapsody™ Express Single-Cell Analysis system for sequencing on Illumina sequencers.
mRNA Targeted and BD® AbSeq Library Preparation Protocol>
mRNA Targeted, BD® AbSeq, and Sample Tag Library Preparation Protocol>
This protocol provides instructions on creating single cell mRNA, Sample Tag, and BD® AbSeq libraries with the BD Rhapsody™ Single-Cell Analysis system or the BD Rhapsody™ Express Single-Cell Analysis system for sequencing on Illumina sequencers.
VDJ Library Preparation Protocolsequencing Library Preparation Protocols
VDJ CDR3 Library Preparation Protocol with Targeted assay
VDJ CDR3 Library Preparation Protocol (Human)>
This protocol provides instructions on creating a single-cell mRNA library with the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina® sequencers. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by generating sequencer-ready libraries specific to the CDR3-specific antigen binding regions of B and T cells.
VDJ CDR3 and BD AbSeq Library Preparation Protocol (Human)>
This protocol provides instructions on creating single-cell mRNA and BD Abseq libraries with the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina® sequencers. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by generating sequencer-ready libraries specific to the CDR3-specific antigen binding regions of B and T cells.
VDJ CDR3 and Sample Tag Library Preparation Protocol (Human)>
This protocol provides instructions on creating single-cell mRNA and Sample Tag libraries with the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina® sequencers. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by generating sequencer-ready libraries specific to the CDR3-specific antigen binding regions of B and T cells.
VDJ CDR3, Sample Tag, and BD AbSeq Library Preparation Protocol (Human)>
This protocol provides instructions on creating single-cell mRNA, Sample Tag, and BD AbSeq libraries with the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina® sequencers. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by generating sequencer-ready libraries specific to the CDR3-specific antigen binding regions of B and T cells for human samples.
VDJ CDR3 Library Preparation Protocol (Mouse)>
This protocol provides instructions on creating a single-cell mRNA library with the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina® sequencers. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by generating sequencer-ready libraries specific to the CDR3-specific antigen binding regions of B and T cells for mouse samples.
VDJ CDR3 and Sample Tag Library Preparation Protocol (Mouse)>
This protocol provides instructions on creating single-cell mRNA and Sample Tag libraries with the BD Rhapsody™ Single-Cell Analysis System or the BD Rhapsody™ Express Single-Cell Analysis System for sequencing on Illumina® sequencers. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by generating sequencer-ready libraries specific to the CDR3-specific antigen binding regions of B and T cells for mouse samples.
TCR/BCR Full Length Library Preparation Protocol with WTA assay (Human)
Human TCR/BCR Full Length and mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol>
Human TCR/BCR Full Length, mRNA Whole Transcriptome Analysis (WTA), and Sample Tag Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome and protein analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina-compatible single-cell barcoded mRNA, TCR, and BCR libraries.
Human TCR/BCR Full Length, mRNA Whole Transcriptome Analysis (WTA), and BD® AbSeq Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina-compatible single-cell barcoded mRNA, TCR, and BCR libraries. The addition of a sample-tag library enables demultiplexing of samples that were pooled before capture on the BD Rhapsody™ system.
This protocol enables high-throughput single-cell transcriptome and protein analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina-compatible single-cell barcoded mRNA, TCR, and BCR libraries. The addition of a sample-tag library enables demultiplexing of samples that were pooled before capture on the BD Rhapsody™ system.
TCR/BCR Full Length Library Preparation Protocol with Targeted assay (Human)
Human TCR/BCR Full Length and Targeted mRNA Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina-compatible single-cell barcoded mRNA, TCR, and BCR libraries.
Human TCR/BCR Full Length, Targeted mRNA, and Sample Tag Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina-compatible single-cell barcoded mRNA, TCR, and BCR libraries. The addition of a sample-tag library enables demultiplexing of samples that were pooled before capture on the BD Rhapsody™ system.
Human TCR/BCR Full Length, Targeted mRNA, and BD® AbSeq Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome and protein analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina-compatible single-cell barcoded mRNA, TCR, and BCR libraries.
Human TCR/BCR Full Length, Targeted mRNA, BD® AbSeq, and Sample Tag Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome and protein analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina-compatible single-cell barcoded mRNA, TCR, and BCR libraries. The addition of a sample-tag library enables demultiplexing of samples that were pooled before capture on the BD Rhapsody™ system.
TCR/BCR Full Length Library Preparation Protocol with Targeted assay (Mouse)
Mouse TCR/BCR Full Length and Targeted mRNA Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina compatible single-cell barcoded mRNA, TCR, and BCR libraries.
Mouse TCR/BCR Full Length, Targeted mRNA, and Sample Tag Library Preparation Protocol>
Mouse TCR/BCR Full Length, mRNA Whole Transcriptome Analysis (WTA), and BD® AbSeq Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina compatible single-cell barcoded mRNA, TCR, and BCR libraries.
Mouse TCR/BCR Full Length, mRNA Whole Transcriptome Analysis (WTA), BD® AbSeq, and Sample Tag Library Preparation Protocol>
TCR/BCR Full Length Library Preparation Protocol with WTA assay (Mouse)
Mouse TCR/BCR Full Length and mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol>
This protocol enables high-throughput single-cell transcriptome analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing instructions for amplifying Illumina compatible single-cell barcoded mRNA, TCR, and BCR libraries.
Mouse TCR/BCR Full Length, mRNA Whole Transcriptome Analysis (WTA), and Sample Tag Library Preparation Protocol>
Mouse TCR/BCR Full Length, Targeted mRNA, and BD® AbSeq Library Preparation Protocol>
Mouse TCR/BCR Full Length, Targeted mRNA, BD® AbSeq, and Sample Tag Library Preparation Protocol>
VDJ Full Length Library Preparation Protocol
dCODE Dextramer® (RiO) Library Preparation Protocol>
This protocol provides instructions on creating dCODE Dextramer® with or without BD® AbSeq Assay and targeted mRNA single cell libraries with the BD Rhapsody™ Express Single-Cell Analysis System and BD Rhapsody™ Single-Cell Analysis System, followed by sequencing on Illumina sequencers. This protocol is an addendum to mRNA Targeted and BD® AbSeq Assay library preparation with the BD Rhapsody™ Targeted mRNA and AbSeq Amplification Kit and is used to obtain dCODE Dextramer® libraries alongside targeted mRNA and optional BD® AbSeq Assay libraries.
Bioinformatics Guides
BD Single Cell Genomics Bioinformatics Handbook>
This handbook is a comprehensive reference to help you prepare and analyze single-cell libraries with the BD Rhapsody™ Single-Cell Analysis system or the BD Rhapsody™ Express Single-Cell Analysis system. Major aspects of the BD® Single-Cell Multiomics bioinformatics workflow are covered. This reference explains the BD single-cell multiomics sequencing analysis algorithms to deepen your understanding of how single-cell mRNA and protein (AbSeq) expression profiles are generated. In addition, the handbook defines every analysis metric.
BD Single Cell Genomics Analysis Setup User Guide>
This guide provides detailed instructions on how to set up and run the BD Rhapsody™ Analysis pipelines on the Seven Bridges Genomics platform or on a local installation.
How to Operate the BD Rhapsody™ Targeted Analysis VDJ CDR3 Pipeline>
This technical bulletin is a supplement to the BD® Single-Cell Multiomics Bioinformatics Handbook and provides a step-by-step instructions on how to operate the BD Rhapsody Targeted Analysis VDJ CDR3 Pipeline.
SeqGeq™ Documentation>
SeqGeq was built by and for scientists, with powerful data reduction tools to help you discover, cluster, and subset populations and relationships. It also includes visualization tools—including heat mapping and easy-to-use report editors—to help you communicate your results. This site provides high-level descriptions of some of SeqGeq’s core functions.
Additional Resources
Flow Cytometry Analysis of BD™ AbSeq Ab-Oligo and Sample Tag Expression>
The purpose of this protocol is to provide detailed instructions on how to use single-color flow cytometry to analyze expression of oligonucleotide-conjugated antibodies with a poly(A) tail. This protocol allows rapid analysis of the expression of protein targets in the sample of interest when labelling cells with Sample Tags from the BD™ Single-Cell Multiplexing Kit or with BD AbSeq Ab-Oligos. This protocol is used prior to cDNA Synthesis
