How do I design custom probes for a Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) for multiplexed samples experiment?

Question: How do I design custom probes for a Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) for multiplexed samples experiment?

Answer:  Fixed RNA Human/Mouse Transcriptome Probe kits contain 4 or 16 probe sets, where each probe set includes a Probe Barcode that enables sample multiplexing and downstream demultiplexing. 

To design custom probes for use in a multiplex experiment, the Probe Barcode portion of the RHS probe (8 bases, gray box in Table 1) will need to be replaced with the corresponding Probe Barcode sequence listed in Table 2. Note that the RHS probes must be designed and synthesized for each Probe Barcode to be used in an experiment. For example, if running a 4-plex experiment, the RHS probe will need to be ordered with one of each of the following four probe barcodes - BC001, BC002, BC003 and BC004. 

When adding custom probes to the hybridization reaction, the Probe Barcode on the custom probe must match the Probe Barcode for the WTA Probes. For example, when setting up a hybridization reaction using WTA Probes BC003, be sure to use the custom RHS probe that includes BC003.

Table 1: Sequences for Single Cell Gene Expression Flex probes.

Table showing the structure of probe sequences for Single Cell Gene Expression Flex. The LHS probe contains the pRead 2S and a 25-base sequence that is the reverse-complement of the mRNA being targeted. The RHS probe contains a 5’ phosphate (/5Phos/), a 25-base sequence that is the reverse-complement of the mRNA being targeted, Constant Sequence, NN, Probe Barcode and pCS1. The NN in the RHS probe is included to recover sequencing quality after reading the Constant Sequence before reading the Probe Barcode.

Each probe in the probe pair represents 25 bps of homology to the target transcript. The target_LHS and target_RHS parts of each probe contain unique 25 bp sequences targeting the transcript of interest. Note that target_LHS and target_RHS should be the reverse-complement of the mRNA sequence being targeted. The Probe Barcode should be replaced with the corresponding Probe Barcode sequence listed in Table 2. For detailed design considerations, please see Custom Probe Design for Visium Spatial Gene Expression and Chromium Single Cell Gene Expression Flex (CG000621). 

Table 2: Recommended Probe Barcode sequences*

* Note that the Probe Barcode sequences in the WTA probe sets are actually a mix of four distinct barcode sequences, which ensures balanced base composition during sequencing. For the purposes of a Custom Probe spike-in experiment, a single Probe Barcode sequence from each mixture is sufficient. For simplicity, the sequence beginning with A from each mixture is recommended. 

While no impact on assay performance is anticipated, the use of custom probes in these assays is not officially supported by 10x Genomics. 10x Genomics cannot guarantee that custom probes will yield data comparable to that from the whole transcriptome panel.  The additional guidance and resources that have been provided in this article are intended to help enable these experiments to improve customer success.

Products: Single Cell Gene Expression Flex, Fixed RNA Profiling