Questions & Answers
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SupportSingle Cell Gene Expression Flex
Fixed 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
- What workflow modifications are required when performing CRISPR Screening with the Single Cell Gene Expression Flex assay?
- How do I pool custom probes for a Single Cell Gene Expression Flex + CRISPR experiment?
- How do I design custom probes for CRISPR Guide Capture for use with the Single Cell Gene Expression Flex Assay?
- 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
- Can dissociated FFPE samples be flow sorted for use with the Fixed RNA Profiling assay?
- Does the Sample Preparation from FFPE Tissue Sections for Chromium Fixed RNA Profiling (CG000632) Demonstrated Protocol produce a suspension of single nuclei, single whole cells or a mixture of both?
- Can I use Flow Sorting with fixed whole blood samples?
- 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?
GEM Generation & Barcoding
Library Construction
Sequencing
- What sequencing configuration is required for antibody libraries derived from using Proteintech Group (PTG) antibodies mixed with Biolegend antibodies, generated from multiplexing experiments using Single Cell Gene Expression Flex?
- What sequencing configuration is required for Proteintech Group (PTG)-derived antibody libraries generated from multiplexing experiments using Single Cell Gene Expression Flex?
- Why was the percentage of PhiX increased for Multiplexed Fixed RNA Profiling libraries sequenced on NovaSeq 6000?
- 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?
Software
- Cell Ranger detected a lower number of cells than expected in my FFPE samples with the Fixed RNA Profiling (Flex) assay. Can I change cell calling parameters to recover more cells?
- Why do you recommend a T nucleotide at the 25th position of custom probes in the Technical Note for custom probe design for Flex and Visium assays?
- How do I analyze CRISPR screening + Single Cell Gene Expression Flex data in Cell Ranger?
- Is trajectory analysis compatible with the Fixed RNA Profiling (Flex) assay?
- How can I get FASTQ files by sample for a multiplexed FRP library?
- How does Fixed RNA Profiling (Flex) data compare to 3' Gene Expression (GEX) data?