Questions & Answers
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SupportFixed RNA Profiling
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)?
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)?
GEM Generation & Barcoding
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?