I only want to check the protein expression level of my samples. I know mRNA will be captured on the bead, but is it necessary to prepare an mRNA library and sequence it as well along with the AbSeq library?

Yes, currently the pipeline requires mRNA reads for cell calling. To ensure that the pipeline can distinguish live cells from dead cells and debris, prepare a shallow (2,000 reads/cell) mRNA library. If an mRNA library is not added, the bioinformatics pipeline will crash at the cell calling step.

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. See technical bulletin Co-staining cells with fluorescent antibodies and BD Rhapsody™ Antibodies for more information.

Can I use the same antibody clone during co-staining?

If it is necessary for your experiment, we recommend co-staining at a 1:1 molar ratio. See technical bulletin Co-staining cells with fluorescent antibodies and BD Rhapsody™ Antibodies for more information.

What is the concentration of BD^® AbSeq Antibodies?

We provide all BD^® AbSeq Ab-Oligos in test size. Each test size is 2 μL; however, the concentration of antibodies may vary. The concentration depends on the identity of the marker type, density of the targets, affinity of the clone, and other factors, so it will differ depending on the antibody specificity. The tubes are packaged according to the titration data to ensure optimal signal-to-noise and performance for each clone.

Can I use antibodies other than the off-the-shelf BD^® AbSeq Reagents?

Yes, with BD^® AbSeq Custom Antibodies, we offer custom conjugation of an AbSeq-qualified oligonucleotide barcode to both antibodies provided by customers and for other BD antibody clones. Contact your regional sales representative to start an order.

What is the minimum and maximum number of BD^® AbSeq Ab-Oligos used in an assay?

There is no minimum BD^® AbSeq Antibody requirement for an assay. The maximum number of BD^® AbSeq Antibodies that can be used is 100. We offer a protocol that can support pooling of antibodies for up to 100-plex AbSeq experiments.

How can I get the .FASTA reference file for my BD^® AbSeq Panel?

Refer to the BD Single-Cell Multiomics Analysis Setup User Guide or use our online BD^® AbSeq Panel generator.

Can I pool the BD^® AbSeq Library with VDJ libraries to sequence them together?

We cannot guarantee an accurate data output ratio when combining the VDJ and AbSeq libraries. We recommend sequencing AbSeq libraries separately in a PE75 cycle sequencing setup as the small AbSeq fragments will outcompete VDJ libraries. If it is necessary to run the AbSeq libraries with the VDJ library, we recommend using a unique indexing reverse primer for the AbSeq libraries so that the AbSeq reads can be trimmed before running the primary analysis pipeline.

Can we multiplex samples that have a varying number of antibodies per sample?

Yes, internally, we have tested a 10-plex Ab-oligo multiplexed with a 40-plex sample and saw minimal residual jumping. Ensure that each sample is washed well for best results.