Reagents

• Is Direct Target Enrichment recommended if the 5' Gene Expression Library is not needed?
• Why do I need an extra kit if I want Gene Expression in addition to V(D)J?
• Can I study T and/or B cell immune repertoires and gene expression profiles from the same cells?
• Can the Single Cell V(D)J kit be used to amplify non-human T or B cell receptors?

Sample Prep - Cells

• 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?

Post GEM-RT Cleanup & cDNA Amplification

• Why is the distribution of fragments after amplification different between T and B cells?
• Why is the annealing temperature and number of enrichment cycles different for T and B cells?
• How do I design a custom targeted assay for Single Cell V(D)J?
• Why are there two target enrichment steps in the V(D)J workflow?

Library Construction

• I have <50 ng cDNA. Can I still make a 5' Gene Expression library?

Sequencing

• Is there a common 5’ UTR captured during V(D)J enrichment?
• Should I adjust the targeted sequencing depth if I did not enrich for T or B cells?
• Can I sequence V(D)J-enriched libraries from T and B cells on the same lane?
• Can I sequence V(D)J and 5' Gene Expression libraries on the same lane?
• Alternative sequencing formats for Single Cell V(D)J libraries
• What Illumina sequencers are supported for Single Cell V(D)J libraries?

Software

• Can I identify differentially expressed genes between cells in two different clonotypes?
• Is the amino acid sequence for the full V(D)J contig available?
• Are the V(D)J and gene expression references interchangeable?
• Will the V(D)J pipeline work without a reference?
• Why is the same TRB chain paired with TRA in clonotype1 and alone in clonotype2?
• Why is the D gene annotation missing in several TCR beta chains?

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