General

• What Fixed RNA Profiling applications are compatible with TotalSeq™️ antibodies?
• When should I start to incorporate the thermal cycler protocol adjustment (updating Pre-Amplification annealing temperature)?
• What happens if I forget to update my thermal cycler program for Pre-Amplification and I perform Barcode Oligo Capture protocols?
• Why is the annealing temperature during Pre-Amplification updated from 67°C to 63°C across all Fixed RNA Profiling protocols?
• Why isn’t TotalSeq™️-B compatible with Fixed RNA Profiling for Multiplexed Samples?
• What 10x kits are required for performing Fixed RNA Profiling with Feature Barcode technology for Protein using Barcode Oligo Capture (TotalSeq™️-C compatible)?

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Custom Probe Design

• How do I design custom probes to detect fluorescent reporters EGFP and/or RFP?
• How do I order and use custom probes in a Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) experiment?
• How do I design custom probes for a Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) for multiplexed samples experiment?

Sample Preparation

• What QC is available before, during, and after running the Chromium Gene Expression Flex assay with FFPE samples?
• Can I use OCT embedded fresh frozen tissue with the Flex/Fixed RNA Profiling assay?
• Can formaldehyde from alternative vendors be used for sample fixation in the Fixed RNA Profiling Assay?
• Can I use formalin fixed tissue as input for the Gene Expression Flex assay?
• Can an FFPE tissue slide be used as input for the Isolation of Cells from FFPE Tissue Sections Demonstrated Protocol (CG000632)?
• Can I use Xylene alternatives for Tissue Deparaffinization during the Isolation of Cells from FFPE Tissue Sections Demonstrated Protocol (CG000632)?

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GEM Generation & Barcoding

• How should I proceed with my Fixed RNA Profiling samples if I experience a clog or wetting failure?
• Why is GEM storage at -20°C not a validated stopping point in the Fixed RNA Profiling assay?

Library Construction

• Why does my Fixed RNA Profiling library look unexpected or have low yield?
• What is the expected size and concentration of Fixed RNA Profiling Libraries?
• Why do I need a different sample index plate (Dual Index Kit TT) to make Fixed RNA Profiling Feature Barcode libraries?
• What dual index plate is compatible with the Chromium Fixed RNA Profiling?

Sequencing

• Why was the percentage of PhiX for NovaSeq increased for Multiplexed Fixed RNA Profiling libraries sequenced on NovaSeq?
• Why are the "Reads Mapped Confidently to the Probe Set" or "Reads Mapped Confidently to the Filtered Probe Set" metrics in my Fixed RNA Profiling web summary file low?
• What sequencing parameters should be used for Fixed RNA Profiling gene expression libraries?
• Can I pool Fixed RNA Profiling gene expression libraries with other 10x Genomics libraries for sequencing?
• What Illumina sequencers are supported for Fixed RNA Profiling libraries?

Software

• How does Fixed RNA Profiling (Flex) data compare to 3' Gene Expression (GEX) data?
• Is it expected to see a significant background signal from genomic DNA in Chromium Fixed RNA Profiling data?
• What does the term fold of coverage mean in the probe-based assays?
• Is it possible to integrate fixed RNA profiling data with 3' or 5' gene expression data?
• Can I use PDX/xenograft tissue with Fixed RNA Profiling?
• Does Cell Ranger normalize UMI counts based on the coverage of genes in Fixed RNA Profiling?

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