Questions & Answers
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SupportSingle Cell Immune Profiling
V(D)J repertoire of T and B cells integrated with 5' Gene Expression and Antigen Specificity
Sample Prep - Cells
- Can PBMCs be cryopreserved in whole blood without isolation for 5’ single cell sequencing?
- Is Single Cell Immune Profiling compatible with nuclei?
- Recommended control Human B cells for V(D)J assay
- Recommended T and B cell enrichment protocols
- What are recommended control Human T cells for V(D)J assay?
Feature Barcode - Cell Surface Protein
- Can I perform Cell Hashing in a GEM-X Universal 5’ Gene Expression v3 workflow?
- Can I perform cell hashing in Next GEM Universal 3’ or 5’ workflows?
- Can I salvage my experiment if I did not use the recommended Feature cDNA primers for Cell Surface Protein during cDNA amplification?
- What are the additional peaks in my Cell Surface Protein library?
- What Demonstrated Protocols for sample preparation are compatible with 5' Feature Barcoding?
- Why do I need a different sample index plate to make 5' Feature Barcode libraries?
Feature Barcode - 5' CRISPR Screening
- Can I target higher cell numbers when performing Single Cell 5’ CRISPR experiments?
- Are MilliporeSigma CRISPR dual guide Lentiviral Vectors validated by 10x Genomics?
- What sequencing length should I use for Read 2 of my 5’ CRISPR library?
- How to decide if the 5’ CRISPR assay or the 3’ CRISPR assay best fit my experiment?
- Why does my 5’ CRISPR Screening library trace have additional peaks?
- Is 5’ CRISPR compatible with my guide of interest?
Feature Barcode - Barcode Enabled Antigen Mapping (BEAM)
- What options are available for antigen specificity studies with the Chromium Single Cell Immune Profiling assay?
- Can I titrate the ratio of biotinylated antigen to BEAM Conjugate to improve the detection of antigen-positive cells in a BEAM-Ab experiment?
- Can I use two fluorophores to identify antigen-positive cells in a BEAM experiment?
- How can I reduce background PE signal for the BEAM-Ab Negative Control Assembly?
- Which antibodies should I include in my fluorescent antibody panel for BEAM flow sorting?
- Can I use more than one MHC allele in a BEAM experiment?
Feature Barcode - Dextramer
- What are the differences between the protocols for Cell Labeling with dCODE™ Dextramer® Reagents from 10x Genomics and Immudex?
- What factors should I consider when planning my antigen specificity experiment using dCODE™ Dextramer® from Immudex?
- How can I combine dCODE™ Dextramer® staining and cell surface protein staining in the same workflow?
High Throughput Single Cell Immune Profiling
Reagents
Reagents for Single Cell 5' VDJ Application.
- What is the poly-dT primer sequence in the GEM-X Single Cell 5’ v3 assay?
- Which reagent kits are compatible with 5’ v3?
- When do I need an additional Library Construction Kit (PN-1000190) to run my experiment with Next GEM 5’ v2 reagent kits?
- Why are the primers designed near the 5' end of the C-region in the V(D)J assay?
- Can I detect T cells with gamma-delta chains in my V(D)J data?
- Why do I need an extra kit if I want Gene Expression and/or Feature Barcoding in addition to V(D)J?
GEM Generation & Barcoding
Post GEM-RT Cleanup & cDNA Amplification
- How can I optimize V(D)J primer design for my non-human/non-mouse species of interest?
- Which genes does each V(D)J-specific primer map to?
- What is the difference between V(D)J Enrichment for Human and Mouse samples?
- Why is the distribution of fragments after amplification different between T and B cells?
- How do I design a custom PCR-based target enrichment assay for Single Cell V(D)J?
- Why are there two target enrichment steps in the V(D)J workflow?
Library Construction
Sequencing
- Is it expected to see low abundance of Ts in cycles 1 and 10 of Read 1 when sequencing 5’ v3 pools?
- What sequencing configuration is needed for Chromium GEM-X Immune Profiling 5’ v3?
- What is the limit of detection for a specific clonotype using the Single Cell Immune Profiling assay?
- Can I sequence my V(D)J libraries in a different sequencing configuration than the ones recommended in the user guide?
- When do I need to sequence my V(D)J-enriched libraries more deeply?
- Is there a common 5’ UTR captured during V(D)J enrichment?
Software
- Web summary metrics for V(D)J Troubleshooting
- How to calculate paired clonotype diversity
- Can I analyze BEAM-Ab and BEAM-T data at the same time? (BEAM = Barcode Enabled Antigen Mapping)
- What are the major updates in Cell Ranger v7.1 that impacts V(D)J data?
- Building a custom reference for V(D)J using IMGT tool
- Loupe Browser investigation for GEX + V(D)J data for differences in cell numbers