CG000621 | Rev CTECHNICAL NOTE

Custom Probe Design for Visium Spatial Gene Expression
and Chromium Single Cell Gene Expression Flex
Introduction

Contents

10x Genomics Visium Spatial Gene Expression
technology, including Visium CytAssist-enabledprobe-based applications, provides spatialtranscriptomic insights by analyzing mRNA intissue sections derived from fixed tissue
samples. Chromium Single Cell Gene Expression
Flex offers comprehensive, scalable solutions tomeasure gene expression in fixed samples.

2		 Visium Spatial Gene Expression
3
3
3
4
5
7

Probe Design
Ordering Custom Probes
Using Custom Probes
Analysis
Data Highlight
Appendix A

10 Chromium Single Cell Gene Expression Flex
For these assays, 10x Genomics provides a predesigned whole transcriptome panel of probes
for target hybridization. Custom probes may
be designed for use with either assay using theguidance provided in this document. While no
impact on assay performance is anticipated,
the use of custom probes in these assays is notsupported or validated by 10x Genomics.
10x Genomics cannot guarantee that custom
probes will yield data comparable to that fromthe whole transcriptome panel.
This Technical Note provides guidance fordesigning and using custom probes, includingprobe pooling and dilution, for Visium Spatial
Gene Expression for FFPE, Visium CytAssistenabled applications, and Chromium Single
Cell Gene Expression Flex (Fixed RNA
Profiling) singleplexed and multiplexed
experiments. The document also includes data
highlighting custom probe specificity.
Additional optimization may be required.
Performing a pilot experiment with these
unsupported workflow modifications is
recommended prior to larger studies.

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12
12
13
13
16

Probe Design
Ordering Custom Probes
Using Custom Probes
Analysis
Data Highlight
Appendix B

19 Conclusion
19 References

Technical Note | Custom Probe Design

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Visium Spatial
Custom Probe

Visium Spatial Gene Expression
Probe Design

Species
Mouse

5'R

ead
2S

LHS
3'

RHS

A 3'
AAA
AAA
A
A
A

Probe Set Version

Direct Placement

Visium Mouse
Transcriptome Kit v1

CytAssist-enabled
Direct Placement

Human
CytAssist-enabled

5'mRNA Target Site

Assay

Visium Human
Transcriptome Kit v1
Visium Human
Transcriptome Kit v2

Table 2. Probe panels used for either Direct Placement or
CytAssist-enabled assays.

Probe Hybridization
3'
AAA
AAA
A
A
AA

5'R

ead
2S

3'

mRNA Target Site

5'

Figure 1. Probe design for the Visium Spatial Gene Expression
assay. The left hand side (LHS) probe contains a partial Read2S as well as a sequence that is reverse complement of thetarget site. The right hand side (RHS) probe contains aphosphate on the 5' base for ligation, sequences reverse
complementary to the target, and a poly(A) tail.

The following guidance applies to both the Visium
Spatial Gene Expression for FFPE (Direct
Placement) and Visium CytAssist Spatial Gene
Expression (CytAssist-enabled) assays.
An overview of the 10x Genomics probe
configuration for Visium Spatial Gene Expression isprovided in Figure 1. The sequences for these probe
designs are provided in Table 1. The probe sets
used for either assay are described in Table 2.

The Visium Human Transcriptome Kit v2 contains
a panel with three pairs of probes for each target
mRNA, with each probe containing 25 bp sequencethat is reverse complement of the target mRNA
sequence. Each probe is referred to as the left handside (LHS) or right hand side (RHS) probe. The
Visium Mouse and Human Transcriptome Kit v1
contain panels with one pair of probes for eachtarget mRNA
When designing custom probes for Visium Spatial
Gene Expression for FFPE, consider the following:• GC content should be between 44 − 72% for each
25 bp probe half.
• Avoid homopolymer repeats.
• Avoid overlap with annotated repeat or low
complexity sequences.
• If possible, design probes for coding regions ofmRNA as opposed to untranslated regions.

Visium Spatial Gene Expression for FFPE Probe Sequence
LHS Probe5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’

RHS Probe/5Phos/-target_RHS-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’

Table 1. Probe sequences for Visium Spatial Gene Expression probe-based applications. The LHS and RHS probes include target_
LHS and target_RHS parts respectively that each contains unique 25 bp sequences homologous to the target transcript. The
target sequence of RHS probes is followed with a series of thirty adenines.

10xgenomics.com

2Technical Note | Custom Probe Design• Avoid common single nucleotide polymorphisms(SNPs) and potential mismatches at the ligation
junction. Refer to the UCSC Genome Browser andthe Single Nucleotide Polymorphism Database(dbSNP). If avoiding SNPs is not possible, SNPs
and mismatches should be at least four bp awayfrom the ligation junction.
• If probes can bind to sequences other than thetarget mRNA sequence, an off-target signal may
be observed. To check for off-target homology,align the probe sequence to the referencetranscriptome using the Basic Local Alignment
Search Tool (BLAST). Matches to off-target genes
should have at least five mismatches in at least
one of the LHS or RHS probes to prevent efficienthybridization.
• Designing three probe pairs per target mRNA
is recommended, especially for low expressing
genes. However, if the gene is not long enough or
there are not enough specific 50 bp regions, fewer
than three probe pairs is acceptable.
• Probe pairs should not overlap to avoid
competition between probes for the same bindingsite in the target RNA.
• Add new probe sequences to the probe set
reference CSV file. Refer to the Analysis section
for more information.

Ordering Custom Probes
Custom probes can be ordered from any
oligonucleotide synthesis provider. 10x Genomics
has tested custom probes in various formats
available from IDT, including DNA Oligos (standard
desalted), Ultramer DNA Oligonucleotides, andoPool Oligo Pools. In limited testing, comparable
results were observed with all formats.

Key Guidelines• Probes should go through standard desalting.
• No HPLC purification is required.
• Probes should be supplied in IDTE (or low EDTATE Buffer).
• RHS probes must be 5' phosphorylated.
• Ordering the custom probes as an oPool ispreferred as it simplifies probe pooling anddilution upstream of the Probe Hybridization
step (see Appendix A).
• LHS and RHS probes can be combined in thesame oPool or ordered as separate pools.

Using Custom Probes
To use custom probes, prepare a combined spike-in
pool (LHS and RHS) containing 24 nM of eachprobe in nuclease-free water. For example, aspike-in pool with 9 probe pairs would contain
24 nM of each of the 9 LHS and 9 RHS probes(432 nM total probe).
Add 10 μl of the spike-in pool (LHS and RHS
combined) to FFPE Hyb Buffer and the 10x
Genomics probes, as shown in Table 3, to generate
the Modified Probe Hybridization Mix. The
Modified Probe Hybridization Mix will replace theoriginal Probe Hybridization Mix in the Probe
Hybridization step of Visium Spatial Gene
Expression for FFPE Reagent Kits User Guide(CG000407) and Visium CytAssist Spatial Gene
Expression for FFPE Reagent Kits User Guide(CG000495).

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3

Visium Spatial
Custom Probe• The 25th nucleotide of the probe (3' most nucleotideof the LHS probe) must be a T. The opposiing
nucleotide in the target RNA must be an A.

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Technical Note | Custom Probe Design

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Modified Probe
Hybridization Mix
Add in order listed
FFPE Hyb Buffer
RHS Probes
Human WTOR
Mouse WTLHS Probes
Human WTOR
Mouse WT
Custom Probes, eachprobe at 24 nM (LHS
and RHS combined)

10x PN

1X (µl)200042370
102000657/ 2000449
2000455
102000658/ 2000450
2000456-

Total

Visium Spatial
Custom ProbeIf performing the CytAssist-enabled assay andworking with 11 mm Capture Areas, double allvolumes in the Modified Probe Hybridization Mix,for a total volume of 200 µl.

the default files.
• For more information, consult "Creating a
Reference Package with spaceranger mkref"
page in the Spatial Gene Expression section
on the 10x Genomics Support website.

Probe Set Reference CSV• Find the appropriate probe set CSV on the
Descriptions of Probe Set Reference CSV andSupporting Files page in the Spatial Gene
Expression for FFPE section of the 10x Genomics
Support website.
• Update the appropriate probe set CSV byappending the new custom probe information,
depending on the probe set:
• Human or Mouse WT Probes v1:

10
200

Table 3. Modified Probe Hybridization Mix for use in the
Visium Spatial Gene Expression assay.

Analysis
The use of custom probes requires the following filemodifications for successful Space Ranger analysis:

Genome Reference• The following steps for modifying genomereference are required if the custom probes
are targeting genes that are not already in the
prebuilt reference. Update the gene annotation
file (GTF) with new targets using a text editor.
• Follow the existing format of Space Ranger
GTF.
• Ensure the gene name is unique.
• Modify the genome reference in FASTA format
that contains additional contigs for new targets.
• Generate a new reference using Space Ranger
mkref, which uses the modified GTF and FASTA
files. This build will be used for data analysis
using Space Ranger.

• If new genes are added and a new genomereference is created using the mkref pipeline,
the #reference_genome and the #reference_version in the header of the new probe set
CSV file should be modified to match thename and version of the genome reference
used for analysis.
• gene_id: the ID of the mRNA target (anyidentifier)• probe_seq: combined LHS and RHS sequence
trimmed to not include any adaptor, R2, orpoly(A) sequences. Target mRNA sequence
only.
• probe_id: pipe-separated gene_id|gene_name|7 character hash (any combination ofletters and numbers)• included: TRUE (will include in Space Ranger
analysis)• Human Probes v2 (same as above, with one
additional edit)• region: spliced or unspliced• spliced: the combined LHS and RHS
sequence spans a splice junction• unspliced: the combined LHS and RHS
sequence does not span a splice junction.
For example, the sequence sits entirely
within a single exon of the target gene.

• Name the new reference and new probe CSV
files so that they can be distinguished from10xgenomics.com

4Technical Note | Custom Probe Design

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Visium Spatial
Custom Probe

Data Highlight
Custom probe pairs were designed to detect EGFP
(Enhanced green fluorescent protein) and mRFP
(monomeric red fluorescent protein, referred to as
RFP in this document) reporter genes in samples
processed as per the Visium CytAssist Spatial Gene
Expression (CG000495) workflow. The probes were
designed based on reference sequences from
Addgene (EGFP, https://www.addgene.org/13031/;
RFP, https://www.addgene.org/13032/).

Probe Design
The target sequence included in the custom probes
is a reverse complement of the common EGFP and
RFP sequences being detected in this experiment.
The LHS probes are listed in Table 4 and the RHS
probes are listed in Table 5. If using the listed
sequences, confirm that the EGFP or RFP
constructs used in an experiment include thebinding site for these probes.

Custom LHS Probes for Visium Gene Expression Assay
Probe
Configuration

5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’

EGFP-LHS-1

CCTTGGCACCCGAGAATTCCAggtagtggtcggcgagctgcacgct

EGFP-LHS-2

CCTTGGCACCCGAGAATTCCAagggtgtcgccctcgaacttcacct

EGFP-LHS-3

CCTTGGCACCCGAGAATTCCAatggtgcgctcctggacgtagcctt

RFP-LHS-1

CCTTGGCACCCGAGAATTCCAtcggtcttgtaggcgccgggcagct

RFP-LHS-2

CCTTGGCACCCGAGAATTCCAaagttggtgccgcgcagcttcacct

RFP-LHS-3

CCTTGGCACCCGAGAATTCCAcctcgatctcgaactcgtggccgtt

Table 4. LHS probe

sequences for detection of
EGFP and RFP reporter genes.

Custom RHS Probes for Visium Gene Expression Assay
Probe
Configuration/5Phos/-target_RHS-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’

EGFP-RHS-1/5Phos/gccgtcctcgatgttgtggcggatc-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAEGFP-RHS-2/5Phos/cggcgcgggtcttgtagttgccgtc-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAEGFP-RHS-3/5Phos/cgggcatggcggacttgaagaagtc-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

RFP-RHS-1/5Phos/gcacgggcttcttggccatgtaggt-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

RFP-RHS-2/5Phos/tgtagatgaactcgccgtcctgcag-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

RFP-RHS-3/5Phos/cacggagccctccatgcgcaccttg-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Table 5. RHS probe sequences for detection of EGFP and RFP reporter genes.

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5Technical Note | Custom Probe Design

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Visium Spatial
Custom Probe

Probe Dilutions
Visium Spatial: Std. Desalt. Probe Dilution Overview

Custom probes used in this study were ordered as
standard desalted probes (100 µM stock) that werediluted as illustrated in the overview (see Appendix A
for additional guidance, including dilution of oPool
Oligo Pools).

Resuspended Stock(100 µM/probe)

LHS

RHS

1

2

Interm. Pool
(480 nM/probe)

Working Stock(24 nM/probe)Mod. Hybrid.
Mix3

Add 10 µl
Working Stock1232.4 µl each
5 µl each (Interm.)(100 µM stock) + 95 µl nuclease-free+ IDTE pH8 for total
water
500 µlVisium Gene Expression for FFPE Experiment Overview & Results
A.

B.

C.

Probe Counts

Log2 Exp
EGFP

EGFP

RFP

EGFP + RFP

EGFP

Log2 Exp
EGFP

0.0

Log2 Exp
11.0
mRFP1

Control

Log2 Exp
EGFP

500

0.0

RFP

Log2 Exp
mRFP1

5 mm

EGFP

RFP EGFP + RFP

10.0

5 mm

Log2 Exp
mRFP1

0.0

0.0

10.0

0.0

5 mm

0.0

0.0
Log2 ExpmRFP1

0.0
Log2 RFP
10.0

5 mm
5 mm

Control

0.0

mRFP1

0.0
0.0

10.0

0

11.0

10.0

5 mm

5 mm

EGFP

Log2 EGFP
11.0

Log2 Exp
11.0

10.0

1000

Log2 Exp

11.0

11.0

2000
1500

Log2 Exp
EGFP

5 mm

0.0

Figure 2. Custom probe specificity in detection of EGFP and RFP in a Visium Gene Expression experiment. In the experiment
5 mm

5 mm

performed, all 4 cell pellets (GFP, RFP, GFP/RFP, neg. control) were embedded in a single FFPE block as a 2x2 array, sectioned onto
a slide, then incubated with the modified hybridization mix, containing both the EGFP and RFP probes (A) and processed andanalyzed as per the Visium CytAssist Spatial Gene Expression (CG000495) workflow. EGFP is detected only in cells expressing
EGFP but not in RFP-expressing cells. Similarly, RFP is only detected in cells expressing RFP but not in EGFP-expressing cells
demonstrating the specificity of the custom probes (B-C).
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6Technical Note | Custom Probe Design

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Visium Spatial
Custom Probe

Appendix A: Visium Spatial Custom
Probe Pooling & Dilution
Custom probe pooling and dilution, upstream of
the Probe Hybridization step, is dependent on theformat in which the custom probe is acquired andthe number of probes being pooled. Example
dilutions for oPools, standard desalted, andUltramer custom probes are provided here.

oPool Oligo Pools
For combining fewer than 30 oPools in a single
spike-in pool, follow the steps described below. If
combining more than 30 oPools,
contact support@10xgenomics.com.
Visium Spatial: oPool Dilution Overview
Resuspended Stock(800 nM/probe)

Working Stock(24 nM/probe)Mod. Hybrid.
Mix

Add 10 µl
Working Stock
3 µl resuspended stock+ 97 µl nuclease-freewater

a. Resuspend stock:
Resuspend oPool Oligos in IDTE (10 mM Tris,
0.1 mM EDTA, pH 8.0) for a stock concentrationof 800 nM/probe. Store resuspended oligos at-20°C.

b. Prepare working stock:
Using the resuspended stock, prepare thespike-in pool working stock containing 24 nM
each of the LHS and RHS probes.

If the spike-in pool contains fewer than 34 probes,
the working stock (24 nM/probe) should be
prepared fresh before use and any remaining
solution should be discarded.
If the spike-in pool contains 34 or more probes,
the working stock (24 nM/probe) may be preparedahead of time and stored at -20°C.
Spike-in Pool Working Stock(24 nM/probe)

Volume (µl)

Nuclease-free water

97

Resuspended Stock*(800 nM/probe)

3

Total

100

*If adding multiple resuspended oPool stocks, add 3 µlfrom each oPool stock and reduce the volume of nucleasefree water proportionally.
c. Pipette mix 15X (pipette set to 90 µl), centrifuge
briefly.
d. Add 10 µl spike-in pool working stock to the
Modified Probe Hybridization Mix (see Table 3).

Resuspension volume is based only on the oPool
scale and not on the number of oligos in the oPool.
To determine resuspension volume, first convert
the oPool scale (provided in pmol/oligo) to nmol
per oligo and then calculate the volume of IDTE
needed for 800 nM/oligo concentration.
Example: Centrifuge oPool tube (50 pmol/oligo scale)briefly, add 62.5 µl IDTE (pH 8.0), and resuspend to aconcentration of 800 nM/oligo.

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7Technical Note | Custom Probe Design

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Visium Spatial
Custom Probe

Standard Desalted or Ultramer
When ordering probes synthesized as individualoligos in tubes or plates, the recommended dilution
scheme depends on the number of total custom
probes being used in the experiment.

c. Vortex 30 sec, centrifuge briefly. Theintermediate stock may be stored at -20°C.
d. Prepare working stock: Using the intermediatepool, prepare a spike-in pool working stock.

If the spike-in pool contains fewer than 34 probes,
the working stock (24 nM/probe) should be
prepared fresh before use and any remaining
solution should be discarded. If the spike-in poolcontains 34 or more probes, the working stock(24 nM/probe) may be prepared ahead of time andstored at -20°C.

Visium Spatial: Std. Desalt. Probe Dilution Overview
Resuspended Stock(100 µM/probe)

LHS

RHS

1

2

Interm. Pool
(480 nM/probe)

Working Stock(24 nM/probe)Mod. Hybrid.
Mix3

Add 10 µl
Working Stock1

2

3

Spike-in Pool Working Stock(24 nM/probe)

2.4 µl each
5 µl each (Interm.)(100 µM stock) + 95 µl nuclease-free+ 485.6 µl IDTE pH8
water

For �200 Total Custom Probes(�100 LHS probes + �100 RHS probes)
a. Resuspend stock:
Resuspend each oligo in IDTE (10 mM Tris,
0.1 mM EDTA, pH 8.0) for a stock concentrationof 100 µM. Store resuspended stock at -20°C.
b. Prepare intermediate pool:
Using the resuspended stock, prepare theintermediate pool by combining 2.4 µl fromeach 100 µM resuspended probe in IDTE
(10 mM Tris, 0.1 mM EDTA, pH 8.0) for a total
volume of 500 µl.

Volume (µl)

Nuclease-free water

95

Intermediate Pool*(480 nM/probe)

5

Total

100

e. Pipette mix 15X (pipette set to 90 µl), centrifuge
briefly.
f.

Add 10 µl spike-in pool working stock to the
Modified Probe Hybridization mix (Table 3).

Example: intermediate pool containing a totalof 6 custom probes
Intermediate Pool
480 nM/probe (100 µM stock)

Volume (µl)

IDTE (pH 8.0)
LHS Custom Probe 1
LHS Custom Probe 2
LHS Custom Probe 3
RHS Custom Probe 1
RHS Custom Probe 2
RHS Custom Probe 3

485.6
2.4
2.4
2.4
2.4
2.4
2.4

Total

500

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8Technical Note | Custom Probe Design

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Visium Spatial
Custom Probe

For 200–4,160 Total Custom Probes(�2,080 LHS probes + �2,080 RHS probes)

For >4,160 Total Custom Probes
Contact support@10xgenomics.com

a. Resuspend stock:
Resuspend each oligo in IDTE (10 mM Tris, 0.1
mM EDTA, pH 8.0) for a stock concentration of
100 µM. Store resuspended stock at -20°C.
b. Prepare working stock:
Using the resuspended stock, prepare a spike-in
pool working stock for a total volume of
10,000 µl.
Example: pool for 300 custom probes or 150
custom probe pairs
Spike-in Pool Working
Stock (100 µM stock)

Volume (µl)

Nuclease-free water

9,280

LHS Custom Probes 1-150

360 (2.4 µl each X 150)

RHS Custom Probes 1-150

360 (2.4 µl each X 150)

Total

10,000c. Vortex 30 sec, centrifuge briefly.
d. Add 10 µl spike-in pool working stock to the
Modified Probe Hybridization mix (Table 3).

Spike-in pool working stock may be preparedahead and aliquots can be stored at -20°C tominimize freeze-thaw.

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9Technical Note | Custom Probe Design

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Chromium Single Cell Gene Expression Flex
10x Genomics probe panels consist of three probe
pairs for most target mRNAs, with each probe
containing appropriate handle sequences and a
25 bp sequence that is the reverse complement of
the target mRNA. Each probe is referred to as theleft hand side (LHS) or right hand side (RHS) probe.
An overview of 10x Genomics probe configurationfor Single Cell Gene Expression Flex is provided in
Figure 3 along with the accompanying probe
sequences for singleplex and multiplex experiments
in Table 6.

Figure 3. Probe design for Chromium Single Cell Gene

Expression Flex. The left hand side (LHS) probe contains a partial
Read 2S (pRead 2S) as well as a 25 bp sequence that is reverse
complement of the target site. The right hand side (RHS) probe
contains a phosphate on the 5' base for ligation, sequences
reverse complementary to the target, Constant Sequence, Probe
Barcode, and a partial capture sequence 1 (pCS1).
The following guidance outlines how to design
custom probes for both singleplex and multiplexed
experiments using the Chromium Single Cell Gene
Expression Flex (Fixed RNA Profiling) assay.

When designing probes for Chromium Flex
experiments, the configuration presented in Table 6
is strongly preferred. Alternatively, a RHS probe
configuration where the Constant Sequence and NN
are omitted may be used to reduce synthesis cost.
However, when using this alternate RHS probe
configuration, the sequencing read parameters must
be changed to Read 1–48 cycles, i7 index–10 cycles,
i5 index–10 cycles, Read 2–50 cycles. If theseparameters are not used, the Probe Barcode will not
be detected correctly by Cell Ranger.

Single Cell Gene Expression Flex Custom Probe Sequence
Singleplex Probe
LHS Probe

RHS Probe5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’/5Phos/-target_RHS-ACGCGGTTAGCACGTA-NN-ACTTTAGG-CGGTCCTAGCAA-3’
Constant Sequence

Probe Barcode

Multiplex Probe
LHS Probe

RHS Probe5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’/5Phos/-target_RHS-ACGCGGTTAGCACGTA-NN-XXXXXXXX-CGGTCCTAGCAA-3’
Constant Sequence

Probe Barcode

Table 6. Single Cell Gene Expression Flex probe configuration with sequences. Each probe in the probe pair represents 25 bp sequences
homologous to the target transcript. The LHS probe sequence is the same for both singleplex and multiplex probes. The RHS probe forsingleplex experiments includes Probe Barcode BC001 sequence ACTTTAGG while for multiplex experiments, each of the RHS probes
must be designed and synthesized with a unique Probe Barcode BC001-BC016 (sequences listed in Table 7). All RHS probes also include
an NN sequence to recover sequencing quality after reading the Constant Sequence before reading the Probe Barcode.
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10

Chromium Flex
Custom Probe

Probe Design

Technical Note | Custom Probe Design

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For singleplex experiments, the Probe Barcode
BC001 sequence "ACTTTAGG" is included in the
RHS probe (Table 6).

Probe Barcode in RHS probe(for multiplexing)

Sequence

BC001
BC002
BC003
BC004
BC005
BC006
BC007
BC008
BC009
BC010
BC011
BC012
BC013
BC014
BC015
BC016

ACTTTAGG
AACGGGAAAGTAGGCT
ATGTTGACACAGACCT
ATCCCAAC
AAGTAGAG
AGCTGTGAACAGTCTG
AGTGAGTG
AGAGGCAAACTACTCAATACGTCAATCATGTG
AACGCCGAATTCGGTT

Table 7. Unique Probe Barcode sequences for integrating in

the RHS probe for multiplexing experiments using the Single
Cell Gene Expression Flex assay.
The Probe Barcode sequences in the WTA probe sets are a mix
of four distinct barcode sequences, which ensures balanced
base composition during sequencing. For the purposes of acustom probe spike-in experiment, a single Probe Barcode
sequence from each mixture is sufficient. For simplicity, thesequence beginning with A from each mixture isrecommended.

WTA Probe

Custom Probe

BC001

BC001

BC003

BC003

BC001

BC002

BC003

BC004

Chromium Flex
Custom Probe

For multiplex experiments, each RHS probe must
be designed and synthesized with a unique eightbase Probe Barcode (BC001-BC016; sequences
listed in Table 7). During the downstream Probe
Hybridization step, when adding custom probes tothe hybridization mix, the Probe Barcode on thecustom probe must match the Probe Barcode of thecorresponding Human/Mouse WTA Probe added tothe mix. See illustrated examples of correct andincorrect Probe Barcode pairings in Table 8.

Pairing

Table 8. Examples of correct and incorrect Probe Barcode
pairings between WTA and custom probes for multiplexing
experiments.

When designing custom probes for either singleplex
or multiplex experiments, consider the following:• GC content should be between 44 − 72% for each
25 bp probe half.
• Avoid homopolymer repeats.
• Avoid overlap with annotated repeat or low
complexity sequences.
• If possible, design probes for coding regions ofmRNA as opposed to untranslated regions.
• The 25th nucleotide of the probe (3' most nucleotideof the LHS probe) must be a T. The opposing
nucleotide in the target RNA must be an A.
• Avoid common single nucleotide polymorphisms(SNPs) and potential mismatches at the ligation
junction. Refer to the UCSC Genome Browser andthe Single Nucleotide Polymorphism Database(dbSNP). If avoiding SNPs is not possible, SNPs
and mismatches should be at least four bp awayfrom the ligation junction.
• If probes can bind to sequences other than thetarget mRNA sequence, an off-target signal may
be observed. To check for off-target homology,align the probe sequence to the referencetranscriptome using the Basic Local Alignment
Search Tool (BLAST). Matches to off-target genes
should have at least five mismatches in at least
one of the LHS or RHS probes to prevent efficienthybridization.

10xgenomics.com

11Technical Note | Custom Probe Design• Designing three probe pairs per target mRNA
is recommended, especially for low expressing
genes. However, if the gene is not long enough or
there aren't enough specific 50 bp regions, fewer
than three probe pairs is acceptable.
• Probe pairs should not overlap to avoid competition between probes for the same binding site in
the target RNA.

Ordering Custom Probes
Custom probes can be ordered from any
oligonucleotide synthesis provider. 10x Genomics
has tested custom probes in various formats
available from IDT, including DNA oligos (standard
desalted), Ultramer DNA Oligonucleotides, andoPool Oligo Pools. In limited testing, comparable
results were observed with all formats.

Key Guidelines• Probes should go through standard desalting.
• No HPLC purification is required.
• Probes should be resuspended in IDTE (or low
EDTA TE Buffer).
• RHS probes must be 5' phosphorylated.
• Ordering the custom probes as an oPool is preferred as it simplifies probe pooling and dilution
upstream of the Probe Hybridization step (seeAppendix B).
• LHS and RHS probes can be combined in thesame oPools or ordered as separate pools.
• When ordering oPools for multiplexing
experiments, RHS probes with different Probe
Barcodes must be ordered as separate pools.
LHS probes may be ordered as a separate oPool
or included with each of the oPools for thedifferently barcoded RHS probes.

Using Custom Probes
To use custom probes, prepare a spike-in poolcontaining 40 nM of each probe in nuclease-freewater. For example, a spike-in pool with 9 probe
pairs would contain 40 nM of each of the 9 LHS
probes and 9 RHS probes (720 nM total probe).
For multiplexed samples, all the RHS probes withina pool should have the same Probe Barcode. For
example, when performing a multiplex experimentusing four Probe Barcodes, four spike-in pools, one
for each Probe Barcode, should be prepared.
Recommended dilution of custom probes upstreamof probe hybridization is dependent on the orderingformat. Example dilutions for oPools, standard
desalted, and Ultramer custom probes are provided
in Appendix B.
5 μl of the custom probe spike-in pool (LHS andRHS probes combined) are added to the sample
after the 10x Genomics Human/Mouse WTA Probes
are added to the resuspended cell pellet as shownin Table 9.
Modified Probe
Hybridization Mix
Add in order listed
Hyb Buffer
Enhancer
Human WTA Probes
OR
Mouse WTA Probes
Custom Probes, eachprobe at 40 nM (LHS
and RHS combined)

10x PN

1X (µl)2000483
2000482
2000495-2000510

70
10
20

2000703-2000718
-

5

Total

105

Table 9. Modified Probe Hybridization Mix for use in the
Chromium Single Cell Gene Expression Flex assay.

When adding custom probes to the Modified Probe
Hybridization Mix, the Probe Barcode for the spike-in
custom probe pool must match the Probe Barcode for
the WTA Probes, as illustrated in Table 8.

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12

Chromium Flex
Custom Probe• Add new probe sequences to the probe set reference CSV file. Refer to the Analysis section for
more information.

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Technical Note | Custom Probe Design

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Analysis
The use of custom probes requires the following filemodifications for successful Cell Ranger (v7.2 orlater) analysis:

Probe Set Reference CSV• Update the appropriate probe set reference CSV
file by appending the new custom probe information in the following columns:• If new genes are added and a new genomereference is created using the mkref pipeline,
the #reference_genome and the #reference_version in the header of the new probe set CSV
file should be modified to match the nameand version of the genome reference used foranalysis.

Data Highlight
Custom probe pairs were designed to detect EGFP
(Enhanced green fluorescent protein) and mRFP
(monomeric red fluorescent protein, referred to as
RFP in this document) reporter genes in cell lines.
Samples were fixed and processed as per the Single
Cell Gene Expression Flex singleplex (CG000691)
and multiplex (CG000527) workflows (seeReferences). The probes were designed based onreference sequences from Addgene (EGFP, https://www.addgene.org/13031/; RFP, https://www.
addgene.org/13032/).

• probe_seq: combined LHS and RHS sequence
trimmed of any adaptor, R2, or partial capture sequences. Target mRNA sequence only.
• probe_id: Pipe-separated gene_id|gene_name|7 character hash (any combination ofletters and numbers)

Chromium Flex
Custom Probe• Find the appropriate probe set CSV on the
Descriptions of Probe Set Reference CSV andSupporting Files page in the Fixed RNA Profiling
section of the 10x Genomics Support website.

• gene_id: the ID of the mRNA target (anyidentifier)• included: TRUE (will include in Cell Ranger
analysis)• region: spliced or unspliced• spliced: the combined LHS and RHS
sequence spans a splice junction• unspliced: the combined LHS and RHS
sequence does not span a splice junction.
For example, the sequence sits entirely
within a single exon of the target gene.

Probe Design
The target sequence included in the custom probes
is a reverse complement of the common EGFP and
RFP sequences being detected in this experiment.
The LHS probes are listed in Table 10 and the RHS
probes are listed in Table 11. If using the listed
sequences, confirm that the EGFP or RFP
constructs used in your experiment include thebinding site for these probes.

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13Technical Note | Custom Probe Design

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Table 10. LHS probe

Custom LHS Probes for Single Cell Gene Expression Flex Assay
5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’

EGFP-LHS-1

CCTTGGCACCCGAGAATTCCAggtagtggtcggcgagctgcacgct

EGFP-LHS-2

CCTTGGCACCCGAGAATTCCAagggtgtcgccctcgaacttcacct

EGFP-LHS-3

CCTTGGCACCCGAGAATTCCAatggtgcgctcctggacgtagcctt

RFP-LHS-1

CCTTGGCACCCGAGAATTCCAtcggtcttgtaggcgccgggcagct

RFP-LHS-2

CCTTGGCACCCGAGAATTCCAaagttggtgccgcgcagcttcacct

RFP-LHS-3

CCTTGGCACCCGAGAATTCCAcctcgatctcgaactcgtggccgtt

Chromium Flex
Custom Probe

Probe
Configuration

sequences for detection of
EGFP and RFP reporter genes.
These LHS probes may bepaired with RHS probes (Probe
Barcode BC001) for singleplex
experiments or with RHS
probes (Probe Barcodes
BC001-BC016 listed in Table 7)
for multiplex experiments.

Custom RHS Probes for Single Cell Gene Expression Flex Assay
Probe
Configuration/5Phos/-target_RHS-ACGCGGTTAGCACGTA-NN-ACTTTAGG-CGGTCCTAGCAA-3’

EGFP-RHS-1/5Phos/gccgtcctcgatgttgtggcggatcACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAAEGFP-RHS-2/5Phos/cggcgcgggtcttgtagttgccgtcACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAAEGFP-RHS-3/5Phos/cgggcatggcggacttgaagaagtcACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA

RFP-RHS-1/5Phos/gcacgggcttcttggccatgtaggtACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA

RFP-RHS-2/5Phos/tgtagatgaactcgccgtcctgcagACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA

RFP-RHS-3/5Phos/cacggagccctccatgcgcaccttgACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA

Table 11. RHS probe sequences for detection of EGFP and RFP reporter genes. These RHS probes containing Probe Barcode

BC001 are for a singleplex experiment. For multiplex experiments, each of the RHS probes must be designed and synthesized witha unique Probe Barcode BC001-BC0016 (sequences listed in Table 7) as described in the Probe Design section.

Probe Dilutions
Custom probes used in this study were ordered as
standard desalted probes (100 µM stock) and werediluted as illustrated in the overview (seeAppendix B for additional guidance, including
dilution of oPool Oligo Pools). When preparing thedilutions for a multiplexing experiment, only RHS
probes with the same probe barcode were added toeach spike-in pool.

Chromium Flex: Std. Desalt. Probe Dilution Overview
Resuspended Stock(100 µM/probe)

LHS
RHS

(same
Probe
Barcode)1

2

Interm. Pool
(800 nM/probe)

Working Stock(40 nM/probe)Mod. Hybrid.
Mix3

Add 5 µl
Working Stock12

3

2 µl each
2 µl each (Interm.)(100 µM stock) + 38 µl nuclease-free+ IDTE pH8 for total
water
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14Technical Note | Custom Probe Design

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Chromium Flex Singleplex Experiment Overview & Results
B.

Library 1

Library 2

Log2 EGFP
12.0

EGFP

A.

Chromium Flex
Custom Probe

0.0

RFP

Log2 RFP
11.0

0.0

Figure 4. Custom probe specificity in detection of EGFP and RFP in a Single Cell Gene Expression Flex singleplex experiment. A
mix of custom probes for detecting EGFP and RFP were added to individual hybridization reactions containing either EGFP- or
RFP-expressing cells (A) and processed and analyzed as per the Chromium Fixed RNA Profiling Reagent Kits for Singleplexed
Samples (CG000691) workflow. EGFP is detected only in cells expressing EGFP but not in RFP-expressing cells. Similarly, RFP is
only detected in cells expressing RFP but not in GFP-expressing cells demonstrating the specificity of the custom probes (B).

Chromium Flex Multiplex Experiment Overview & Results
A.

B.

BC001
BC001

BC002

BC003

BC004

BC003

BC002

BC004

GFP
RFP

EGFP
RFP

Figure 5. Custom probe specificity in detection of EGFP and RFP in a Single Cell Gene Expression Flex multiplex experiment. In a

4-sample multiplex experiment, a mix of custom probes for detecting EGFP and RFP were added to individual hybridization
reactions containing either EGFP cells, RFP cells, a 50:50 mix of EGFP:RFP cells, or negative control cells (A). After hybridization,
samples were pooled in equal proportion, washed, and loaded into a single GEM lane and were processed and analyzed as per the
Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples (CG000527) workflow. Using a UMI threshold of 1 or greater,
EGFP is detected only in samples containing EGFP cells (BC001 and BC003) but not in RFP-only (BC002) or in negative control(BC004) cells. Similarly, RFP is only detected in samples expressing the RFP reporter (BC002 and BC003) but not in the EGFP-onlysample or in the negative control (B).

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15Technical Note | Custom Probe Design

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Appendix B: Chromium Flex Custom
Probe Pooling & Dilution

oPool Oligo Pools
For combining fewer than 20 oPools in a single
spike-in pool, follow the steps described below. If
combining more than 20 oPools,
contact support@10xgenomics.com.

Chromium Flex: oPool Dilution Overview
Resuspened Stock Conc.
(800 nM/probe)

Working Stock(40 nM/probe)Mod. Hybrid.
Mix

Add 5 µl
Working Stock

2 µl resuspended stock +38 µl nuclease-free watera. Resuspend stock:
Resuspend oPool Oligos in IDTE (10 mM
Tris, 0.1 mM EDTA, pH 8.0) for a stock
concentration of 800 nM/probe. Store
resuspended oligos at -20°C.

Resuspension volume is based only on the oPool
scale and not on the number of oligos in theoPool. To determine resuspension volume, first
convert the oPool scale (provided in pmol/oligo)
to nmol per oligo and then calculate the volume
of IDTE needed for 800 nM/oligo concentration.
Example: Centrifuge oPool tube (50 pmol/oligo
scale) briefly, add 62.5 µl IDTE (pH 8.0), andresuspend for a concentration of 800 nM/oligo.

b. Prepare working stock:
Using the resuspended stock, prepare thespike-in pool working stock containing 40 nM
each of the LHS and RHS probes.

If the spike-in pool contains fewer than 20 probes,
the working stock (40 nM/probe) should be
prepared fresh before use and any remaining
solution should be discarded.
If the spike-in pool contains more than 20 probes,
the working stock (40 nM/probe) may be preparedahead of time and stored at -20°C
Spike-in Pool Working Stock

Chromium Flex
Custom Probe

Custom probe pooling and dilution, upstream ofthe probe hybridization step, is dependent on theformat in which the custom probe is acquired andthe number of probes being pooled. Example
dilutions for oPools, standard desalted, andUltramer custom probes are provided here.

Volume (µl)

Nuclease-free water

38

Resuspended Stock*(800 nM/probe)

2

Total

40

*If adding multiple resuspended oPool stocks, add 2 µlfrom each oPool stock and reduce the volume of nucleasefree water proportionally.
c. Pipette mix 15X (pipette set to 30 µl), centrifuge
briefly.
d. Add 5 µl spike-in pool working stock to the
Modified Probe Hybridization mix (see Table 9).

All RHS probes in a spike-in pool working stock
should have the same Probe Barcode. For example,
when performing a multiplex experiment using four
Probe Barcodes, four spike-in pools, one for each
Probe Barcode, should be prepared.
When adding custom probes to the Modified
Probe Hybridization Mix, the Probe Barcode for the
spike-in custom probe pool must match the Probe
Barcode of the WTA Probes used in the probehybridization reaction.

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16Technical Note | Custom Probe Design

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Standard Desalted or Ultramer
Example: intermediate pool for Probe Barcode
BC001 containing a total of 6 custom probes

When ordering probes synthesized as individualoligos in tubes or plates, the recommended dilution
scheme depends on the number of total custom
probes being used in the experiment.
Chromium Flex: Std. Desalt. Probe Dilution Overview

LHS
RHS

(same
Probe
Barcode)1

2

Interm. Pool
(800 nM/probe)

Working Stock(40 nM/probe)Mod. Hybrid.
Mix3

Add 5 µl
Working Stock12

3

2 µl each
2 µl each (Interm.)(100 µM stock) + 38 µl nuclease-free+ 238 µl IDTE pH8
water

Volume (µl)

IDTE (pH 8.0)
LHS Custom Probe 1
LHS Custom Probe 2
LHS Custom Probe 3
RHS Custom Probe 1, BC001
RHS Custom Probe 2, BC001
RHS Custom Probe 3, BC001

238
2
2
2
2
2
2

Total

250

Chromium Flex
Custom Probe

Resuspended Stock(100 µM/probe)

Intermediate Pool (BC001)
800 nM/probe (100 µM stock)c. Vortex 30 sec, centrifuge briefly. Theintermediate stock may be stored at -20°C.
For �120 Total Custom Probes(�60 LHS probes + �60 RHS probes)

d. Prepare working stock: Using the intermediatepool, prepare a spike-in pool working stock.

a. Resuspend stock:
Resuspend each oligo in IDTE (10 mM Tris, 0.1
mM EDTA, pH 8.0) for a stock concentration of
100 µM. Store resuspended stock at -20°C.

Spike-in Pool
Working Stock (BC001)
Nuclease-free water38

b. Prepare intermediate pool:
Using the resuspended stock, prepare theintermediate pool by combining 2 µl from each
100 µM resuspended probe in IDTE (10 mM
Tris, 0.1 mM EDTA, pH 8.0) for a total volumeof 250 µl.

Intermediate Pool*(800 nM/probe)

2

Total

40

All of the RHS probes within a pool should havethe same Probe Barcode. For example, when performing a multiplex experiment using four Probe
Barcodes, four intermediate pools, one for each
Probe Barcode, should be prepared.

Volume (µl)

e. Pipette mix 15X (pipette set to 30 µl), centrifuge
briefly.
f.

Add 5 µl spike-in pool working stock to the
Modified Probe Hybridization mix (Table 9).

When adding custom probes to the Modified
Probe Hybridization Mix, the Probe Barcode for the
spike-in custom probe pool must match the Probe
Barcode of the WTA Probes used in the probehybridization reaction.

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17Technical Note | Custom Probe Design

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For 120-2,500 Total Custom Probes(�1,250 LHS probes + �1,250 RHS probes)

For >2500 Total Custom Probes
Contact support@10xgenomics.com

a. Resuspend stock:
Resuspend each oligo in IDTE (10 mM Tris, 0.1
mM EDTA, pH 8.0) for a stock concentration of
100 µM. Store resuspended stock at -20°C.
Chromium Flex
Custom Probeb. Prepare working stock:
Using the resuspended stock, prepare a spike-in
pool working stock for a total volume of
5,000 µl.
Example: pool for Probe Barcode BC001
containing a total of 300 custom probes or 150
custom probe pairs
Spike-in Pool Working Stock(BC001) (100 µM stock)

Volume (µl)

Nuclease-free water

4,400

LHS Custom Probes (1 -150)300(2 µl each X 150)

RHS Custom Probes BC001(1-150)300(2 µl each X 150)

Total

5,000

c. Vortex 30 sec, centrifuge briefly.
d. Add 5 µl spike-in pool working stock to the
Modified Probe Hybridization mix (Table 9).

All RHS probes in a spike-in pool working stock
should have the same Probe Barcode. For example,
when performing a multiplex experiment using four
Probe Barcodes, four spike-in pools, one for each
Probe Barcode, should be prepared.
When adding custom probes to the Modified
Probe Hybridization Mix, the Probe Barcode for the
spike-in custom probe pool must match the Probe
Barcode of the WTA Probes used in the probehybridization reaction.

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18Technical Note | Custom Probe Design

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Conclusion

References

This Technical Note provides guidance on thedesign and use of custom probes with the Visium
Spatial Gene Expression and Chromium Single Cell
Gene Expression Flex (singleplexed and multiplexed)
assays. While no impact on assay performance isanticipated, the use of custom probes in these assays
has not been tested extensively and is not supportedby 10x Genomics. Performing a pilot experimentwith these unsupported workflow modifications is
recommended prior to committing to larger studies.

1.

Visium Spatial Gene Expression for FFPE Reagent
Kits User Guide (CG000407).

2.

Visium CytAssist Spatial Gene Expression Reagent
Kits User Guide (CG000495).

3.

Chromium Fixed RNA Profiling Reagent Kits
User Guide for Singleplexed Samples with Feature
Barcode technology for Protein (CG000477).

4.

Chromium Fixed RNA Profiling Reagent Kits User
Guide for Singleplexed Samples (CG000691).

5.

Chromium Fixed RNA Profiling Reagent Kits
User Guide for Singleplexed Samples with Feature
Barcode technology for Protein using Barcode Oligo
Capture (CG000674).

6.

Chromium Fixed RNA Profiling Reagent Kits User
Guide for Multiplexed Samples (CG000527).

7.

Chromium Fixed RNA Profiling Reagent Kits
User Guide for Multiplexed Samples with Feature
Barcode technology for Protein using Barcode Oligo
Capture (CG000673).

10xgenomics.com

19Technical Note | Custom Probe Design

CG000621 | Rev C

Document Revision Summary
Document Number

CG000621

Title

Custom Probe Design for Visium Spatial Gene Expression and Chromium Single
Cell Gene Expression Flex

Revision
Revision Date

Rev C
October 2023

Specific Changes:
Visium Spatial Updates• Includes Data Highlight section (pages 5-6)
• Includes Appendix A with Custom Probe Pooling & Dilution (pages 7-9)
Chromium Flex Updates• Includes guidance for using custom probes in multiplex experiments (pages 10-12)• Genome Reference guidance removed (page 13)• Includes Data Highlight section (pages 13-15)• Includes Appendix B with Custom Probe Pooling & Dilution (pages 16-18)• Updated References (page 19)

General Changes:
Updated for general minor consistency of language, terms, and format throughout.

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