Question: How do I analyze CRISPR screening + Single Cell Gene Expression Flex data in Cell Ranger?
Answer: The analysis of Singleplex Flex + CRISPR data is enabled in Cell Ranger 7.1 and later.
The analysis of Multiplex Flex + CRISPR data is enabled in Cell Ranger 8.0. For analyzing multiplex experiments, the Multi Config CSV needs to specify probe barcodes for both WTA and CRISPR (see example below). The probe barcodes should be specified as a pair for each sample, e.g. BC001+CR001 for the GEX and CRISPR libraries, respectively. For additional information and examples of Multi Config CSVs for Flex+CRISPR experiments, please see the following Software page: Fixed RNA Profiling with Cell Ranger multi.
Similar to our current on market CRISPR workflows, a feature reference file will be needed to identify the different protospacers. Please see an example below:
[gene-expression]
reference,/path/to/transcriptome
probe-set,/path/to/probe-set.csv
create-bam,false
[libraries]
fastq_id,fastqs,feature_types
frp_gex,/path/to/fastqs,Gene Expression
frp_cr,/path/to/fastqs,CRISPR Guide Capture
[feature]
reference,/path/to/feature_reference.csv
[samples]
sample_id,probe_barcode_ids
sample1,BC001+CR001
sample2,BC002+CR002
sample3,BC003+CR003
sample4,BC004+CR004
Additional Flex + CRISPR Resources:
- For details on how to design custom probes for CRISPR Guide Capture for use with the Flex assay please see: How do I design custom probes for CRISPR Guide Capture for use with the Single Cell Gene Expression Flex Assay?
- For details on workflow modifications required when performing CRISPR screening with the Flex assay please see: What workflow modifications are required when performing CRISPR Screening with the Single Cell Gene Expression Flex assay?
- For details on how to pool custom probe pools for a Flex + CRISPR experiment please see: How do I pool custom probes for a Single Cell Gene Expression Flex + CRISPR experiment?
While no impact on assay performance is anticipated, the use of custom probes in these assays is not officially supported by 10x Genomics. The additional guidance and resources that have been provided in this article are intended to help enable these experiments and improve customer success.
Products: Single Cell Gene Expression Flex, Fixed RNA Profiling