VDJ CDR3 Protocols

The following protocols provide instructions on creating single-cell mRNA 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. To create the library, cDNA of gene targets are encoded on BD Rhapsody™ Cell Capture Beads. This targeted library approach employs a two-step nested amplification, followed by an index PCR step. The generated library can be sequenced on various Illumina sequencers.

BD Rhapsody VDJ protocol is compatible with BD Rhapsody - Targeted, Abseq and SMK protocols either individually or combined.

IMPORTANT: The protocol described herein has not been thoroughly tested or optimized and is provided as is and without warranty. Any use of this protocol is at the risk of the user. BD Biosciences reserves the right to change specifications at any time. Information in the protocol is subject to change without notice. BD Biosciences assumes no responsibility for any errors or omissions. In no event shall BD Biosciences be liable for any damages in connection with or arising from the use of this protocol.

Cell Preparation and Cell Labeling

1. Preparing Single Cell Suspensions

• 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.
• Doc ID: 210964 Rev. 1.0
• Revision date: 07/2018

1. Single Cell Labelling with the BD Single-Cell Multiplexing Kit

• The BD Single-Cell Multiplexing Kit (Cat. No. 633781) utilizes an innovative antibody-oligo technology to provide higher sample throughput for single cell library preparation. Every antibody-oligo in the kit, referred to as a Sample Tag, has a unique sample barcode conjugated to a human universal antibody. Up to 12 samples can be labelled and pooled prior to single cell capture with the BD Rhapsody™ Single-Cell Analysis system or other single cell analysis systems.
• Doc ID: 210970 Rev. 2.0
• Revision date: 02/2019

1. Single Cell Labelling with BD™ AbSeq Ab-Oligos

• This protocol describes use of BD AbSeq Ab-Oligos (antibody-oligonucleotides).
• Doc ID: 214394 Rev. 1.0
• Revision date: 07/2018

1. 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)(1 plex to 40 plex) with the BD® Single-Cell Multiplexing Kit.
• Doc ID: 214419 Rev. 2.0
• Revision date: 02/2019

1. 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.
• Doc ID: 23-22354-00
• Revision date: 05/2020

cDNA Synthesis

1. 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.
• Doc ID: 210967 Rev. 1.0
• Revision date: 07/2018

1. 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.
• Doc ID: 210966 Rev. 1.0
• Revision date: 07/2018

Sequencing Library Preparation

1. 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. 
• Doc ID: 23-22202-00
• Revision date: 01/2020

1. 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. 
• Doc ID: 23-22203-01
• Revision date: 05/2020

1. 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. 
• Doc ID: 23-22205-00
• Revision date: 01/2020

1. 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. 
• Doc ID: 23-22204-01
• Revision date: 05/2020

1. 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.
• Doc ID: 23-22687-00
• Revision date: 05/2020

1. 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. 
• Doc ID: 23-22688-00
• Revision date: 05/2020