Bd rhapsody protocol. Connectivity Software.

Bd rhapsody protocol We highly recommend using the BD Rhapsody scanner (part of this protocol) as it provides quality control checks during the process of sample preparation and cartridge loading. Cell load below or above this recommended range may not be suitable for Use this handy planner to pick the right protocol for your needs BD Rhapsody ™ System TCR/BCR Next, Targeted mRNA, BD ® AbSeq, and Sample Tag Library Preparation Protocol BD Rhapsody™ System TCR/BCR Next and mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol The BD Rhapsody™ HT Xpress is our newest instrument which can run an 8-lane cartridge and offers the same performance as the BD Rhapsody™ Express with a broad range of cell input, from 100–100,000 cells per lane of the cartridge. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by BD Rhapsody™ Protocol Planner; Articles in this section Choose the correct protocol to suit your needs; Choose the correct protocol to suit your needs Updated August 31, 2023 21:29. This protocol provides instructions on creating sing le-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. A63880) BD Rhapsody™ system single-cell labeling with BD® AbSeq Ab-Oligos (41 to 100 plex) 5 Introduction 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. This technical bulletin consolidates the sequences into tables which can be used to identify relevant sequences for specific applications. With scanner : BD Rhapsody™ HT Single-Cell Analysis System Single-Cell Capture and cDNA BD Rhapsody™MultiomicATAC-SeqAmplificationKit BDBiosciences 41928 BD Rhapsody™EnhancedCartridgeReagentKitV3a BDBiosciences 667052 BD Rhapsody™cDNA Kit BDBiosciences 633773 BD Rhapsody™WTA AmplificationKit BD Biosciences 633801 BD Rhapsody™8-LaneCartridge BDBiosciences 666262 Agencourt®AMPure®XPmagneticbeads BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol ; BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol ; BD Single-Cell Genomics Bioinformatics Handbook ; BD Rhapsody™ WTA Amplification Kit Product Insert VDJ CDR3 Demo Data. When enabled, the BD Rhapsody™ Sequence Analysis Pipeline can use the reads from these libraries to assemble contigs corresponding to rearranged TCR and BCR chain Requiredequipment Material Supplier Catalogno. † Completely resuspend the beads by pipet-mixing, then pipet the calculated The BD Rhapsody™BD Rhapsody™ Single-Cell Analysis System enables us to capture multimodal information from thousands of single cells in parallel (“Multiomics”) covering mRNA expression levels, protein expression levels, the immune repertoire for T-cell receptors (TCR) and B-cell receptors (BCR), and the identification of antigen-specific T cells and B cells using Recommendedconsumables Material Supplier Catalogno. Cell Preparation and Separation Reagents . With scanner : BD Rhapsody™ HT Single-Cell Analysis System Single-Cell Capture and cDNA The BD Rhapsody™ WTA Kit is tested and validated to give consistent, high-quality whole transcriptome data across different workflows and users and a wide cell input range. A63880) Protocol for CITE-seq applications BD® OMICS-Guard Buffer allows antibody staining before or after sample preservation. Choose the correct protocol to suit your needs; Sample Prep and Preservation. ® 5® 6 ® ® Rhapsody™ Rhapsody™ Rhapsody™ a. We demonstrate that AbSeq on the BD Rhapsody • Rhapsody Protocol: BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and AbSeq Library Preparation Protocol (23-24118(01)) 6 Mercatelli D, Balboni N, De Giorgio F, Aleo E, Garone C, Giorgi FM. For complete instrument procedures and safety information, see the BD Rhapsody™ Single-Cell 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. . ® ® BD Rhapsody™ system TCR/BCR full length, targeted mRNA, and BD® ® 7 12 ® AbSeq library preparation protocol ® AbSeq library preparation ® 5 BD STOP after the section “Washing the Cell Capture Beads” and follow this protocol from Preparing BD Rhapsody™ Enhanced Cell Capture Beads for TCR/BCR full length, WTA, and Sample Tag library amplification and subsequent steps. This does not reflect the true viability of nuclei loaded but the counts generated are accurate. ideal for routine use with familiar cell systems that do not require visual QC or troubleshooting. How many samples can I multiplex with the BD Rhapsody™ ATAC-Seq standalone or the Multiomic ATAC+mRNA assay? BD Rhapsody Express showing proper cartridge position with 5 mL LoBind Tube for collection of Cell Capture Beads and waste container installed. Details about the individual reagents needed to run the assay are 8 BD Rhapsody system Mouse VDJ CDR3 and Sample Tag library protocol Preparing BD Rhapsody™ Cell Capture Be ads for VDJ library amplification cDNA synthesis and template switching Thaw reagents (except for the enzymes) in the BD Rhapsody cDNA Kit (Cat. This demo data was generated from an experiment using previously frozen resting human PBMCs and enriched human B cells that were multiplexed and stained with BD® AbSeq Immune Discovery Panel+ 4 AbSeq drop-ins and a 15-plex Abseq panel, respectively, whereby ~10,000 cells were captured with the BD Rhapsody™ STAR Protocols is an open access, peer-reviewed journal from Cell Press. Included here are instructions for how to generate BD® AbSeq single-cell libraries and Sample Tag libraries using the BD Rhapsody™ Single-Cell Analysis system, or the BD Rhapsody™ Express Single-Cell Analysis system. Clinical Diagnostics; Clinical Discovery ; Research Reagents; Western Blotting and Molecular Reagents. This enables more precise estimations of cell/bead capture rates, thus allowing optimized sequencing and downstream analysis. You could also buy RNAse inhibitor from our recommended 3rd party vendors NEB and Watchmaker Genomics. Here we describe the complete BD Rhapsody™ Single-Cell Analysis System from the sample preparation including different options for the antibody BD Rhapsody™ system mRNA WTA and AbSeq library preparation protocol 7 Required materials • Exonuclease I-treated BD Rhapsody™ Enhanced Cell Capture Beads containing sample • BD Rhapsody™ WTA Amplification Kit (Cat. e. Technical Notes. † 20,000–1 8BD Rhapsody system VDJ CDR3, Sample Tag, and BD AbSeq library protocol Preparing BD Rhapsody™ Cell Capture Beads for VDJ library amplification cDNA synthesis and template switching Thaw reagents (except for the enzymes) in the BD Rhapsody cDNA Kit (Cat. ® ® BD Rhapsody™ system TCR/BCR full length, targeted mRNA, and BD® ® 7 12 ® AbSeq library preparation protocol ® AbSeq library preparation ® 5 BD 6 BD Rhapsody™ system mRNA targeted and BD® AbSeq protocol Workflow NOTE Univ. A63880) The BD Rhapsody ™ HT Xpress System leverages our proprietary, gentle, robust micro-well based single-cell partitioning technology to perform single cell analysis. BD Rhapsody system mRNA WTA protocol 11 5 (Optional) Subsample the Exonuclease I-treated BD Rhapsody Cell Capture Beads: † Based on the expected number of viable cells captured on beads in the final bead-resuspension volume, determine the volume of beads to subsample for sequencing. 633731) or BD Rhapsody™ Enhanced Cartridge BD Rhapsody™ Single-Cell VDJ CDR3 Assay enables high-throughput detection of VDJ recombination events by generating sequencer-ready Skip to main content. BD disclaims all express and implied warranties, including, but not limited to, merchantability and fitness for use for a particular purpose. A63 80) BD Rhapsody™ system TCR/BCR full length and mRNA WTA library preparation protocol 11 STOPPING POINT: BD Rhapsody™ Enhanced Cell Capture Beads can be stored up to 4 days at 2 °C to 8 °C after template switching. Synthetic poly-A tail on cDNA 3’ end hybridizes to poly-T on its own bead. Read and BD Rhapsody™ System BD® AbSeq Library Preparation Protocol (for AbSeq-based cell calling) Author: BD Biosciences Created Date: 12/14/2022 11:44:36 AM The BD ® Single-Cell Multiplexing Kits allow you to combine and simultaneously process up to 24 different samples in one single-cell multiomics experiment on the BD Rhapsody ™ Single-Cell Analysis System. A63 80) BD Rhapsody™ Single-Cell Analysis System - Cartridge Workflow. In addition to cell surface protein analyses, BD ® AbSeq Antibody-Oligos can now also be used for intracellular CITE-seq. Visual workflow QC for confidence in every experiment >80% cartridge capture rate for certain cell types BD Rhapsody™ System mRNA Targeted Library Preparation Protocol. BD AbSeq Index PCR 7777 BD AbSeq PCR1 LibraryReverse Primer LibraryForwardPrimer AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level Author links open overlay panel Jami R. Doc ID: 23 Sequencing-ready Library Preparation Protocols. Cell load below or above this recommended range The protocol can be downloaded here: (Click here to access the protocol). 1st Stopping Point. The labeling of single cells with universal antibody BD Rhapsody™ TCR/BCR Amplification Kit, Mouse 666282 Companion products BD ® AbSeq Single-Vial Reagents Contact for more info BD® AbSeq Immune Discovery Panel 625970 BD® Immune Response Panel 633750 BD® Human Single-Cell Multiplexing Kit 633781 BD® Mouse Single-Cell Multiplexing Kit 633793 BD® Flex Single-Cell Multiplexing Kit A 633849 BD® Flex BD Rhapsody™ system mRNA WTA, AbSeq, and Sample Tag library preparation protocol 9 Safety information For safety information, see the BD Rhapsody™ Single-Cell Analysis Instrument User Guide (Doc ID 214062) or the BD Rhapsody™ Express Single-Cell Analysis System Instrument User Guide (Doc ID 214063). The BD Rhapsody Scanner uses BD Rhapsody™ Protocol Planner. BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol ; BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol ; BD Single-Cell Genomics Bioinformatics Handbook ; BD Rhapsody™ WTA Amplification Kit Product Insert Can I flow sort my sample before using the BD Rhapsody™ System workflow? Yes, we recommend co-staining cells with fluorescent and oligo nucleotide conjugated antibodies (BD Rhapsody™ Single-Cell Multiplexing Kit or BD ® AbSeq Ab-Oligos) before cell sorting. The sample used for this demo data is freshly thawed frozen PBMCs. The BD Rhapsody™ Single-Cell Multiplexing Kit utilizes an innovative antibody-oligo technology to provide higher sample throughput for single-cell library preparation . Use of other tubes could lead to sub-optimal results. The BD Rhapsody™ Single-Cell Analysis System enables high-throughput capture of multiomic information from single cells. With scanner : BD Rhapsody™ HT Single-Cell Analysis System Single-Cell Capture and cDNA Analytical Workflows for Single-Cell Multiomic Data Using the BD Rhapsody Platform Curr Protoc. The BD Rhapsody™ Scanner reports quality metrics in real-time at each step of your single cell assay from initial cell capture to bead retrieval. Learn more from the BD ® We demonstrate that AbSeq on the BD Rhapsody • Rhapsody Protocol: BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and AbSeq Library Preparation Protocol (23-24118(01)) 6 Mercatelli D, Balboni N, De Giorgio F, Aleo E, Garone C, Giorgi FM. Required materials For a complete list of materials, see instrument user guide. To use the BD Rhapsody™ Single-C ell Analysis system without the BD Rhapsody Scanner, see the BD Rhapsody™ Express Single-Cell Analysis System Instrument User Guide (Doc ID: 214063). Products Instruments Reagents Software & Informatics Solutions Industry The BD Single-Cell Multiplexing Kit (BD Biosciences) was used to combine and load two samples (tumor tissue and corresponding benign tissue) onto a single BD Rhapsody cartridge (BD Biosciences). VDJ CDR3 Protocol(s) Overall Workflow. Bead loading In the protocol sections, Preparing cell capture beads and Loading and washing cell capture beads, use Sample buffer-RI instead In combination with other BD Rhapsody™ assays, optional protocols and products enable the generation of sequencing libraries specific to T- and B-Cell Receptors (TCR and BCR). 633801) • Agencourt® AMPure®XP m ag ne ti cb d s(B k C oul r L f S , . Flow Cytometry Reagents. † BD Rhapsody™ Cartridge Reagent Kit (Cat. It is also intended to provide a method to enable a high-throughput detection of VDJ recombination events by The BD Rhapsody™ HT Xpress is the main hardware for the BD Rhapsody™ protocol. BDRhapsody™Scannera BDBiosciences 633701 BDRhapsody™HTXpressSystema BDBiosciences 700036499 Hemocytometeradaptera BDBiosciences 633703 BDRhapsody™P1200µLPipette-HTXa BDBiosciences 500066148 BDRhapsody™P8xP1200µLPipette-HTXa BDBiosciences 500066280 Finally, our direct comparative analysis corroborates that the BD Rhapsody protocol excels in characterizing cells with low-mRNA content, capturing leukocytes more effectively. Doc ID : 23-22746-00; Revision date: 2020; Enhanced Bead Data Sheet . Safety Use standard laboratory safety protocols. The basic principle is BD Rhapsody™ Enhanced Cell Capture Beads using a random priming approach, followed by an index PCR step. In line with our recent observation in lung cancer BD Rhapsody™ Sequence Analysis Pipeline User's Guide This guide provides detailed instructions on how to set up and run the BD Rhapsody™ Skip to main content . This important benefit of the microwell-based platform is based on a significantly better mRNA capture efficiency. 633774) at For complete instrument procedures, safety information, and workflow, see the BD Rhapsody™ Express Single-Cell Analysis System Instrument User Guide (Doc ID: 214063). Introduction This protocol describes preparation of single cell suspensions of commonly used immune cell types. The BD Rhapsody™ Single-Cell Analysis System consists of two main platforms, the BD Rhapsody™ Scanner and BD Rhapsody™ Express. Pipettes(P10,P20,P200,P1000) Majorsupplier – Low-retention,filteredpipettetips Majorsupplier – 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. section of the BD RhapsodyTM protocol Both the BD® AbSeq and dCODE® amplicons are ~170bp and can not be quantified seperatly at this step. cDNA and LMO libraries were prepared using a custom protocol combining BD Rhapsody's mRNA whole transcriptome analysis (WTA) library preparation The BD Rhapsody™ HT Xpress System provides a broader cell input range (100–440,000 cell per cartridge) on the NEW BD Rhapsody™ 8-Lane Cartridge, enabling capture of more than 320,000 cells per cartridge without compromising performance. BD® AbSeq Enhancers can be added to the BD Fc Block™ Reagent step or BD® AbSeqAb-Oligos(1plexto40plex)Protocol,23-24262orBDRhapsody™SystemSingle-CellLabeling withBD ®AbSeqAb-Oligos(41plexto100plex)Protocol,23-22314,orBDRhapsody™SystemSingle-Cell LabelingwithBD®Single-CellMultiplexingKitandBD®AbSeqAb-Oligos(1plexto40plex)Protocol, 23 6 BD Rhapsody single-cell labeling with BD Single-Cell Multiplexing Kit and BD AbSeq Ab-Oligos (41 to 100 plex) Required materials For a complete list of materials, see the appropriate BD Rhapsody instrument user guide. 16 Place the tube from step 14 Without SMK : BD® AbSeq Library Preparation Protocol. 0; Revision date: 07/2018 Sequencing Library Preparation BD Rhapsody™ VDJ CDR3 Protocol Overview Presentation Slide Deck. 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 8 BD Rhapsody™ system mRNA targeted, sample tag, and BD® AbSeq library preparation protocol Before you begin • Obtain Exonuclease I-treated and inactivated BD Rhapsody™ Enhanced Cell Capture Beads. 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 BD Rhapsody ™ System Single-Cell ATAC‑Seq and mRNA Whole Transcriptomic Analysis (WTA) Library Preparation Protocol; BD Rhapsody™ System Single-Cell ATAC-Seq Library Preparation Protocol; For Research Use Only. Single-Cell Multiomics Help Center BD Biosciences Website Home BD Biosciences; FAQ; Assays; Articles in this section BD Rhapsody™ ATAC-Seq; VDJ CDR3; AbSeq; Single-Cell Multiplexing; BD Rhapsody VDJ protocol is compatible with BD Rhapsody - Targeted, Abseq and SMK protocols either individually or combined. Doc ID: 23-24126(02) Revision date: 11/2022 BD RhapsodyTM Express Single-Cell Analysis system Doc IDs: 214063 and 210967 Kits: 3, 4, 5 BD RhapsodyTM Single-Cell Analysis system Doc IDs: 214062 and 210966 Kits: 3, 4, 5 210966 NO SCANNER SCANNER Library preparation Sequencing and analysis Doc IDs: 47383 and 54169 Whole transcriptome analysis (WTA) only Part Number: 23-21711-00 Kit: 6 Targeted The BD Rhapsody™ WTA Kit is tested and validated to give consistent, high-quality whole transcriptome data across different workflows and users and a wide cell input range. Required materials The scanner has been redesigned to be compatible for use with the BD Rhapsody™ HT Xpress System and BD Rhapsody™ 8-Lane Cartridge for higher-throughput applications, enabling a multisample workflow. This pipeline performs analysis of single-cell multiomic sequence read (FASTQ) data. New synthetic poly-A tail is added on cDNA 3’ BD Rhapsody™ system TCR/BCR full length, targeted mRNA, BD® AbSeq, and sample tag library preparation protocol 9 Procedure Perform the experiment on the BD Rhapsody™ Single-Cell Analysis system following either the: • BD Rhapsody™ Single-Cell Analysis System Instrument User Guide (Doc ID 214062) BD Rhapsody™ system TCR/BCR full length, mRNA WTA, BD® AbSeq, and sample tag library preparation protocol 9 Procedure Perform the experiment on the BD Rhapsody™ Single-Cell Analysis system following either the: • BD Rhapsody™ Single-Cell Analysis System Instrument User Guide (Doc ID 214062) STOP after the section “Washing the Cell Capture Beads” and BD Rhapsody™ system TCR/BCR full length, targeted mRNA, and BD® AbSeq library preparation protocol 11 STOPPING POINT: BD Rhapsody™ Enhanced Cell Capture Beads can be stored up to 4 days at 2 ®. Time considerations # # 6 BD Rhapsody™ system mRNA targeted and BD® AbSeq protocol Workflow NOTE Univ. Visual workflow QC for confidence in every experiment >80% cartridge capture rate for certain cell types ; Gentle settling of cells in microwells, ideal for fragile cells such as neutrophils; Broad range of cell throughput: 100-40,000 cells; Low multiplet BD Rhapsody™ System Whole transcriptome analysis alpha protocol 12/2018 Overview This protocol is intended to provide a method to screen RNA expression of single cells using a 3' whole transcriptome analysis (WTA) approach using both BD-orderable reagents and third-party reagents as outlined in this document. 5-mL tube magnet until the solution is clear ( 1 minute). 2nd Stopping Point. Any use of this protocol is at the risk of the user. a 1,200 rpm and at 25°C for 10 minutes b 1,200 rpm and at 37°C for 15 minutes c 1,200 rpm and at 45°C for 10 minutes d 1,200 rpm and at 55°C for 10 minutes IMPORTANT Set the ramp rates at maximal and set “Time Mode ” to “Temp Control” before the program begins. No. using the BD® AbSeq Immune Discovery Panel (IDP). Doc ID : 23-24228(01) 8 BD Rhapsody system VDJ CDR3, Sample Tag, and BD AbSeq library protocol Preparing BD Rhapsody™ Cell Capture Be ads for VDJ library amplification cDNA synthesis and template switching Thaw reagents (except for the enzymes) in the BD Rhapsody cDNA Kit (Cat. Erickson 1 , Florian Mair 1 , Grace Bugos 1 3 , Jody Martin 2 4 , Aaron J. The BD Rhapsody™ VDJ CDR3 Protocol utilizes the existing BD Rhapsody™ Targeted Kits and the human/mouse immune response primer panel and is designed to work alongside the BD ® AbSeq Assay and BD ® Single-Cell Multiplexing Kit (SMK). In 2022, BD introduced a new version of beads , called Enhanced Beads, which have slightly different oilgo design. Incubate the cell suspension between 20°C and 22°C for 20 min. Single Nuclei Sample Prep Protocols; Single Cell Sample Prep and Preservation(using BD® OMICS-Guard) Protocols The power of Multiomics relies on simultaneously measuring several aspects of single cells, including gene expression and protein abundance, using next generation sequencing (NGS) as a single readout. This guide provides detailed instructions on how to set up and run the BD Rhapsody™ Sequence Analysis Pipeline on the Seven Bridges Genomics platform or on a local server installation. • Thaw reagents in the BD Rhapsody™ Targeted mRNA and AbSeq Amplification Kit (Cat. See our nuclei prep protocols for details. Doc ID : BD-52956; This datasheet talks 8 BD Rhapsody™ system mRNA targeted, sample tag, and BD® AbSeq library preparation protocol Before you begin • Obtain Exonuclease I-treated and inactivated BD Rhapsody™ Enhanced Cell Capture Beads. 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. Note: The BD Rhapsody System will read 100% viability since all nuclei will be positive for DyeCycle Green. Immunoassay Reagents. Preparation Protocol (23-24013) • BD Rhapsody ™ System TCR/BCR Full Length and WTA Library Preparation Protocol (23-24017) • BD Rhapsody™ System Library Preparation Protocol TCR/BCR Full Length, Targeted mRNA, and Sample Tag (23-24014) • BD Rhapsody™ System Library Preparation Protocol TCR/BCR Full Length, mRNA Whole Transcriptome Analysis BD Rhapsody™ Kits and BD® Single-Cell Multiplexing Kits include several primers used in BD Rhapsody Library preparation. In this technical bulletin, we show how isolated nuclei can be captured and amplified on the BD Rhapsody™ Single-Cell 12 BD Rhapsody system mRNA WTA protocol 15 Program the thermomixer. It can be used with or without the BD Rhapsody™ Scanner. Doc ID: 76918, Revision: 4. Time considerations # # Basic Protocol 1: IMAGE ANALYSIS. NOTE Use only the tubes specified in the protocol. The BD Rhapsody™ HT Xpress is a nonelectrical hardware with a magnet and one slider on the front and is mostly used to hold the cartridge in place and to extract the BD Rhapsody™ Enhanced Cell Capture Beads from the cartridge. IMPORTANT: The protocol described herein has not been thoroughly tested or optimized and is provided as is and without warranty. BDRhapsody™Scannera BDBiosciences 633701 BDRhapsody™ExpressInstrumenta BDBiosciences 633702 HemocytometerAdaptera BDBiosciences 633703 BDRhapsody™P1200Mpipettea BDBiosciences 633704 BDRhapsody™P5000Mpipettea BDBiosciences 633705 Largemagneticseparationstand Human TCR/BCR Full Length Demo Data. 11 12 ® tube. • Carefully 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. This protocol provides instructions on creating a single cell mRNA library after cell capture on the BD Rhapsody™ BD Rhapsody™ system mRNA targeted protocol 11 10 Ramp heated lid and heat block of post-amplification thermal cycler to ≤95 °C by starting the thermal cycler program and then pausing it. In this video, we will demonstrate how to use the BD Rhapsody™ Scanner to check the quality of your single cell multiomics experiments. This guide provides instructions on operating the BD Rhapsody™ Scanner, the BD Rhapsody™ Express instrument and supporting materials. Can I use the 100 cycle kit? Yes, our pipeline can accommodate shorter read lengths for WTA or Targeted assays to take the advantage of the 100 cycle kit on the NovaSeq™ Sequencer. If stopping after template switching: • Place the bead suspension on the 1. Doc ID: 214063 Rev. Software & Informatics. For details on the steps of the TCR and BCR analysis or ATAC-Seq analysis, see their dedicated pages. For use with BD Rhapsody™ HT Enhanced Cell Capture Beads V3 (catalog number 667052), do not use with any other bead versions. Sample Collection and Preservation BD Vacutainer® Tubes for cell and biomarker p reservation BD Rhapsody ™ System TCR/BCR Next, Targeted mRNA, BD ® AbSeq, and Sample Tag Library Preparation Protocol BD Rhapsody™ System TCR/BCR Next and mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol 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. Single Nuclei Sample Prep Protocols; Single Cell Sample Prep and This protocol provides instructions on creating a single cell mRNA library after cell capture on the BD RhapsodyTM Single-Cell Analysis system or the BD RhapsodyTM Express Single-Cell This protocol provides instructions on creating single cell whole transcriptome mRNA and AbSeq protein-detection libraries after cell capture on the BD Rhapsody™ Single This protocol enables high-throughput single-cell transcriptome analysis alongside TCR and BCR profiling of individual cells captured on the BD Rhapsody™ system, providing BD Rhapsody™ system TCR/BCR full length and targeted mRNA library preparation protocol 5 Introduction This protocol enables high-throughput single-cell transcriptome analysis alongside Proprietary BDTM Molecular Indexing technology is used to count individual mRNA molecules. Tyznik 2 , Margaret Nakamoto 2 , Stefanie Mortimer 2 , Martin Prlic 1 3 5 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. The BD Rhapsody™ assays are used to Cell capture, cDNA synthesis and exonuclease I treatment were performed using the standard protocols from BD Rhapsody Express Single-Cell Analysis System Instrument User guide (Doc ID 214062). The kits are designed to work with all BD Rhapsody™ Assays and include streamlined informatics tools integrated into the BD Rhapsody ™ Bioinformatics Analysis BD ® AbSeq Antibody-Oligos enable single-cell CITE-seq analyses (simultaneous detection of proteins and mRNA expression) and help deepen your understanding of protein biology to accelerate your single-cell research. Do not proceed to thermal cycling until each tube is gently mixed by pipette to ensure uniform bead suspension. no. This protocol enables users to use cell surface proteins to profile cells independently of mRNA. The first publication (V1 version) is in Nature Methods 16, 75-78 (2019). Both the whole transcriptome mRNA and Sample Tag libraries can be combined together for sequencing on various Illumina sequencers. For a complete cycle of BD Rhapsody. Single-Cell Multiomics Help Center BD Biosciences Website Home BD Biosciences; User Guides and Protocols; Bioinformatics; Articles in this section Bioinformatics; Targeted Reference files 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. 0; Update on: 05/2018; BD Rhapsody™ Single-Cell Analysis System Instrument User Guide. Add 200 μL of BD Stain Buffer to the cell suspension and mix by pipetting . The barcodes are the same, but This protocol covers the following steps: Design of RoCKseq capture sequences and ROIseq primers RoCKseq bead modification on BD Rhapsody beads RoCK and ROI library generation Sequencing of RoCK Rhapsody™System MouseTCR/BCRNext,mRNAWhole TranscriptomeAnalysis(WTA), BD®AbSeq,andSampleTag LibraryPreparationProtocol 23-24519(01) 2024-01 ForResearchUseOnly 23-21340(02) 2022-11 ForResearchUseOnly BDRhapsody™System Single-CellLabelingwith BD®Single-CellMultiplexingKits Protocol. 7 8 ® BD Rhapsody system mRNA WTA and AbSeq library preparation protocol 7 Required materials † Exonuclease I-treated BD Rhapsody Cell Capture Beads containing sample † BD Rhapsody™ WTA Amplification Kit (Cat. The BD Rhapsody™ Express System enables single-cell capture and barcoding of hundreds to thousands of single cells for analysis of genomic and proteomic information, using proprietary, gentle, robust microwell-based single-cell The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. See technical bulletin Co-staining cells with fluorescent antibodies and BD Rhapsody™ Antibodies Explore the BD Rhapsody™ Express Single-Cell Analysis System for single-cell capture and barcoding. The acceptable criteria for valid R1 and R2 reads is 60 bases for R1, the entirety of which should match the cell barcode This section provides an in-depth description of each step in the BD Rhapsody™ Sequence Analysis Pipeline. They use similar split-pool approach to generate their oligos on magnetic beads. A single cell suspension can be loaded into a BD Rhapsody™ Cartridge. 2. BD FACSLink™ BD Rhapsody™ system TCR/BCR full length, targeted mRNA, and sample tag library preparation protocol 9 Procedure Perform the experiment on the BD Rhapsody™ Single-Cell Analysis system following either the: • BD Rhapsody™ Single-Cell Analysis In combination with other BD Rhapsody™ assays, optional protocols and products enable the generation of sequencing single-nuclear ATAC-Seq fragments. BD Rhapsody™ HT Xpress System Instrument User Guide for Human TCR/BCR Next dataset can be found here. 0 out of 0 found this helpful. This protocol provides instructions on creating a single cell whole transcriptome mRNA library after cell capture on the BD RhapsodyTM Single-Cell Analysis system or the BD RhapsodyTM Intended use The BD Rhapsody Single-Cell Analysis system is intended for the preparation of single cell mRNA sequencing libraries. For CITE-seq applications using BD® AbSeq Antibody-Oligos AFTER sample preservation, use of the BD® AbSeq Enhancer Kit (Cat. The fragments are then analyzed to identify nucleosome-free BD Rhapsody™ system mRNA WTA, AbSeq, and Sample Tag library preparation protocol 9 Safety information For safety information, see the BD Rhapsody™ Single-Cell Analysis Instrument User Guide (Doc ID 214062) or the BD Rhapsody™ Express Single-Cell Analysis System Instrument User Guide (Doc ID 214063). The Transcriptome of SH-SY5Y at Single-Cell Resolution: A CITE-seq Data Analysis Workflow. When enabled, the BD Rhapsody™ Sequence Analysis Pipeline can use the reads from these libraries to evaluate open chromatin regions. For complete instrument procedures and safety information, see the BD BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol Author: BD Biosciences Created Date: 11/30/2022 10:12:09 AM All users of the BD Rhapsody™ Express instrument should read this information. 633774) at BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol ; BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol ; BD Single-Cell Genomics Bioinformatics Handbook ; BD Rhapsody™ WTA Amplification Kit Product Insert BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol ; BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol ; BD Single-Cell Genomics Bioinformatics Handbook ; BD Rhapsody™ WTA Amplification Kit Product Insert BD Rhapsody™ VDJ CDR3 Protocol with V1 beads. doi Basic Protocol 1: Image analysis Basic Protocol 2: Sequencing quality control and generation of a gene expression matrix Basic Protocol 3: Gene expression matrix data pre-processing and analysis Basic Protocol 4: Advanced analysis Basic Protocol 5: Proceed to step 6 of the protocol section. BD ® Cytometric Bead Array Solution; BD Immunoassay ELISA Reagents; BD ® ELISPOT BD Rhapsody WTA is a nanowell-based commercial system like Microwell-seq. Note: Smaller contaminating products may be present after PCR1. The libraries were prepared and sequenced according to the BD Rhapsody System TCR/BCR Next, mRNA Whole Transcriptome Analysis (WTA), and BD® AbSeq Library Preparation Protocol (Doc ID 23-24510). ATAC+mRNA : Single-Cell ATAC-Seq and mRNA Whole Transcriptome Analysis Library Preparation Protocol. Every antibody-oligo in a Multiplexing kit, referred to as a Sample Tag, has a unique sample barcode conjugated to a human or BD Rhapsody™ Protocol Planner. 2024 Feb;4(2): e963. Required materials 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. Genomics technical publications are available for download from BD Rhapsody™ Express Single-Cell Analysis System Instrument User Guide (Doc ID: 214063). Doc ID: 23-22951(02) Revision date: 11/2022; 8 lane High Throughput cartridge. For BD Rhapsody™ system TCR/BCR full length, targeted mRNA, and BD® AbSeq library preparation protocol 11 STOPPING POINT: BD Rhapsody™ Enhanced Cell Capture Beads can be stored up to 4 days at 2 ®. We recommend buying additional RNAse inhibitor from BD Biosciences on this link. 633773) at room temperature (15°C to 25°C). The system is capable of barcoding and capturing more than 640,000 single Note: The original BD Rhapsody protocol uses the whole volume of 20 μL of each sample tag-coupled antibody mix for labeling the samples. Connectivity Software. The system is also compatible with the BD Rhapsody™ Scanner for the visual workflow QC with a NEW multisample workflow. Oligo: Universal Oligo; region (dark purple) between universal oligo and poly(dT): cell label and Unique Molecular Identifier. BD AbSeq Index PCR 7777 BD AbSeq PCR1 LibraryReverse Primer LibraryForwardPrimer 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. View Rhapsody™System TCR/BCRNextandmRNAWhole TranscriptomeAnalysis(WTA) LibraryPreparationProtocol 23-24508(01) 2024-01 ForResearchUseOnly 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. Ensure that the intended total cell load is between 7,500–20,000 single cells for this protocol. The supported sequencing libraries are those generated by the BD Rhapsody™ assay kits, including: Whole This protocol describes the preparation of single-cell suspensions of commonly used immune cell types. 23-21338(02)_ Rhapsody_SCA_Workflow-ruo. Not for use in diagnostic or therapeutic procedures. It can also be used in combination with the BD Rhapsody™ Express System and BD Rhapsody™ Single-Lane Cartridge for lower-throughput needs. 23-22946-00. This demo data was generated from an experiment using previously frozen human PBMCs, in which ~8,000 cells were captured with the BD Rhapsody™ Single-Cell Analysis System and processed using the BD Rhapsody™ System TCR/BCR Next and mRNA Whole Transcriptome Analysis (WTA) Library Preparation 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. I want to sequence my BD Rhapsody™ Libraries on a NovaSeq™ Sequencer. Frozen PBMCs were thawed, stained with IDP and loaded onto a BD Rhapsody ™ 8-Lane Cartridge. Here we differentiated Scanner, the BD Rhapsody™ Express instrument and supporting materials. Addition of DNA polymerase and reagents allows copying of new barcodes to cDNA 3’ end (equivalent to mRNA 5’ end) Ability to amplify from the 5’ end of cDNA. This protocol does not describe tissue dissociation. Doc ID : 23-24474(02) Revision date: 08/2024 Requiredequipment Material Supplier Catalogno. This protocol is intended to provide a method to screen RNA expression of single cells using a 3' whole transcriptome analysis The BD Rhapsody™ Single-Cell Analysis System includes the BD Rhapsody™ Scanner and BD Rhapsody™ Express Single-Cell Analysis System. 570750) is highly recommended. The main portion of these steps decribe the WTA mRNA, Targeted mRNA, AbSeq, and Sample Tag processing steps. Interested in demo data for VDJ CDR3 for Enhanced Beads? Please contact us at The BD Rhapsody ™ Scanner can be From reagents required for cell multiplexing and staining to assays and bioinformatics tools, streamlined protocols and technical support, BD provides the tools you need to accelerate your discoveries using single-cell multiomic analysis. 633801) † Agencourt® AMPure® XP magnetic beads (Beckman Coulter Life Sciences, Cat. Keep enzymes at –25°C to –15°C. Cells are entirely contained in the BD RhapsodyTM Cartridge, which is a single-use consumable. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all areas of life, health, earth and physical sciences. BDRhapsody™HTXpressPackagea BDBiosciences 666730 BDRhapsody™P8xP1200µLPipette-HTXb BDBiosciences 500066280 BDRhapsody™P1200µLPipette-HTXc BDBiosciences 500066148 Cellcounter MajorSupplier – BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol ; BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol ; BD Single-Cell Genomics Bioinformatics Handbook ; BD Rhapsody™ WTA Amplification Kit Product Insert ; Multiomics protocols (WTA + AbSeq) BD 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. Sample-tag staining was performed according to the manufacturer's protocol (sample-tag staining at room temperature for 20 min and washing by centrifugation at Requiredequipment Material Supplier Catalogno. 0 10 BD Rhapsody Express Single-Cell Analysis System Instrument User Guide Safety symbols Introduction This topic describes BD Rhapsody system mRNA WTA and AbSeq library preparation protocol 7 Required materials † Exonuclease I-treated BD Rhapsody Cell Capture Beads containing sample † BD Rhapsody™ WTA Amplification Kit (Cat. Performing PCR 2 to amplify dCODE® library from the purified dCODE®/AbSeq fraction from PCR 1 Download scientific diagram | Setting Up the BD Rhapsody Express BD Rhapsody Express showing proper cartridge position with 5 mL LoBind Tube for collection of Cell Capture Beads and waste BD Biosciences has documentation to support many different experimental protocols regarding single-cell multiomics, flow cytometry, cell biology and others. However, using 10 μL yielded comparable results. It is also intended to BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit ; BD Rhapsody™ TCR/BCR Next Multiomic Assays; BD Rhapsody™ Targeted mRNA Kits; BD Rhapsody™ Accessory Kits; Functional Assays. During the workflow, the slider needs to be moved either to “Waste” or to “Beads” as indicated in the protocol. pdf (200 KB) Was this article helpful? Yes No. BD Biosciences reserves the right to BD Rhapsody system mRNA WTA and AbSeq library preparation protocol 7 Required materials † Exonuclease I-treated BD Rhapsody Cell Capture Beads containing sample † BD Rhapsody™ WTA Amplification Kit (Cat. g. For complete instrument procedures and safety information, see the BD Rhapsody™ Single-Cell Analysis System STOP after the section “Washing the Cell Capture Beads” and follow this protocol from Preparing BD Rhapsody™ Enhanced Cell Capture Beads for TCR/BCR full length library amplification and subsequent steps. Each BD ® AbSeq Ab-Oligo Instrument User Guide (Doc ID: 214063) and the BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol (23-21711-00) or the BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol (23-21712-00). BD Rhapsody Express Single-Cell An alysis system are provided for research purposes only. Microscopy and Imaging Reagents. BD Rhapsody™ Protocol Planner. 1. The scanner is an automated microscope equipped with a low-magnification objective that produces batches of images at each single-cell capture step. TM. The data set generated from this protocol can be input BD Rhapsody™ system mRNA WTA library preparation protocol 5 Introduction 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. 0; Revision date: 07/2018 Sequencing Library Preparation 8 BD Rhapsody system VDJ CDR3 and BD AbSeq library protocol Preparing BD Rhapsody™ Cell Capture Be ads for VDJ library amplification cDNA synthesis and template switching Thaw reagents (except for the enzymes) in the BD Rhapsody cDNA Kit (Cat. More Resources Learn more about Proceed to cell loading, bead loading and lysis as outlined in the protocol ‘Single-Cell Capture and cDNA Synthesis with the BD Rhapsody Express Single-Cell Analysis System’ (Doc ID: 23-22952 BD Rhapsody™ system mRNA WTA and AbSeq library preparation protocol 7 Required materials • Exonuclease I-treated BD Rhapsody™ Enhanced Cell Capture Beads containing sample • BD Rhapsody™ WTA Amplification Kit (Cat. BD Rhapsody™EnhancedCartridgeReagentKitV3a BDBiosciences 667052 BD Rhapsody™8-LaneCartridge BDBiosciences 666262 Agencourt ®AMPure®XPmagneticbeads BeckmanCoulter A63880 100%ethylalcohol Majorsupplier – Nuclease-freewater Majorsupplier – N,N-Dimethylformamide MilliporeSigma D4551-250ML DyeCycle™Greenb ThermoFisher Scientific BD Rhapsody™ Express Single-Cell Analysis System; BD Rhapsody™ Scanner; BD Rhapsody™ HT Xpress System; Reagents. For complete instrument procedures and safety information, see the BD Rhapsody™ Single-Cell Analysis System Instrument User Guide (Doc ID: 214062). For complete instrument procedures and safety information, see the BD Rhapsody™ Single-Cell Single Cell Sample Prep and Preservation(using BD® OMICS-Guard) Protocols; Single Nuclei Sample Prep Protocols Updated August 30, 2024 22:19. Doc ID: 210966 Rev. Proceed to spin The BD Rhapsody™ Single-Cell Analysis System enables high-throughput capture of multiomic information from single cells. Full size image. For WTA : Processing nuclei samples on the BD Rhapsody™ Single-Cell Analysis System . f. ~3000 cells were captured using BD Rhapsody Single-Cell Analysis System and sequencing libraries were generated using VDJ CDR3 Library Preparation Protocol (Human) for V1 beads. apcx ixo fhghf qlcfo jychnq qxim oslzxr rqtjav sptrra hlyuc