Laboratory protocol · Step 1
CG000789 · Revision B · Pages 49–53

Step 1: Probe Hybridization

GEM-X Flex multiplexed-sample preparation for Gene Expression and Protein Expression libraries. This protocol begins with labeled, fixed, and quenched samples and ends after probe hybridization.

42°C Hybridization
16–24 h Incubation window
850 × g Sample spin
≤500,000 Maximum input
Protocol summary

Step 1: Probe Hybridization

Prepare Hyb Mix, pellet the fixed samples, assign one WTA Probe Barcode and one Antibody Multiplexing Barcode to each sample, and hybridize at 42°C for 16–24 h.

Input
Labeled, fixed, and quenched cell or nuclei suspension
Output
Samples hybridized with WTA and Antibody Multiplexing Barcodes
Incubation
42°C for 16–24 h with a heated lid and no shaking
Source section
CG000789, Revision B, Step 1, pages 49–53
Step 1 overview

Unique sample → unique probe barcode

Fixed single-cell suspensions receive one WTA Probe Barcode and one Antibody Multiplexing Barcode per sample, followed by overnight hybridization at 42°C.

1 Fixed sample Labeled, fixed, quenched
2 Centrifuge 850 × g · 5 min · 4°C
3 Resuspend 40 µl Hyb Mix
4 Add WTA probe 10 µl · unique per sample
5 Add AB barcode 2 µl · unique per sample
6 Hybridize 42°C · 16–24 h · no shaking
1.0 · Get started

Prepare by thermal state

Warm reagents must remain warm and fully dissolved; barcodes, probes, and the prepared cell suspension remain on ice until the procedure directs otherwise.

Reagents

Part numbers, preparation, and storage from CG000789 Rev B.

Thaw & keep warm
Hyb Buffer B PN 2001312 Thaw at 42°C. Vortex and centrifuge briefly. Keep warm and confirm that no precipitate remains. Do not place the thawed buffer on ice. It may remain at 42°C for up to 1 h. −20°C
Enhancer PN 2000482 Heat at 65°C for 10 min. Vortex and centrifuge briefly. Keep warm and confirm that no precipitate remains. Do not place on ice. It may remain at 42°C for up to 10 min; if precipitation appears after that, reheat at 65°C until fully dissolved. −20°C
Place on ice
Labeled & fixed cell suspension Upstream material Must be appropriately labeled, fixed, quenched, and resuspended in Quenching Buffer B. Use the applicable Demonstrated Protocol selected through the GEM-X Flex Protocol Planner, CG000780. On ice
Antibody Multiplexing Barcode AB001–AB016 PN 2000917–2000932 Thaw on ice. Vortex and centrifuge briefly. −20°C
Human or Mouse WTA Probes BC001–BC016 Human: PN 2001259–2001274
Mouse: PN 2001275–2001290 Thaw on ice. Vortex and centrifuge briefly. −20°C

Equipment required by the procedure

Confirm availability before warming reagents.

Preflight

Resolve inputs before warming

300,000 Default fixed cell or nuclei input when sufficient material is available.
500,000 max Do not exceed 500,000 fixed cells or nuclei in one hybridization reaction.
≤100,000 Recommended for splenocytes, leukocytes, and bone-marrow mononuclear cells because higher loads may slightly reduce data quality.
<50,000 Low input may make it difficult to target the maximum downstream load. At ≤50,000 cells, prefer a 1.5-ml tube when a thermomixer with heated lid is available.
25,000 → ≤25,000 When a cell suspension starts fixation with 25,000 cells, the guide expects no more than 25,000 fixed cells to move forward into hybridization.

Pre-hybridization normalization is optional and design-dependent.

If the same number of cells or nuclei is added to every reaction that will later be pooled, the guide permits pooling without the post-hybridization counting step. This page does not choose that downstream strategy for you.

Guide-linked low-input cautions (pages 33–34)

Using fewer than 25,000 cells may increase the risk of pellet loss, inadequate material for storage or maximum loading, difficult equal-number pooling, difficult counting, and an atypical library trace. The guide suggests careful low-input handling, a swinging-bucket rotor, and leaving up to 15 µl supernatant during this hybridization step to protect recovery.

Hyb Mix · 20% overage

Mix at room temperature

Add components in the listed order. Every batch includes 20% overage. The 2X batch is calculated by doubling the guide’s 1X + 20% volumes. Select a batch to highlight its column.

Hyb Mix volumes with 20 percent overage; the 2X batch is calculated from the CG000789 Revision B 1X recipe
Component 10x PN 1X + 20% 2X + 20% 4X + 20% 16X + 20%
Hyb Buffer B
Add warm; return to 42°C if it appears milky. 		2001312 42.0 µl 84.0 µl 168.0 µl 672.0 µl
Enhancer
Add warm after 65°C heating and precipitate check. 		2000482 6.0 µl 12.0 µl 24.0 µl 96.0 µl
Total — 48.0 µl 96.0 µl 192.0 µl 768.0 µl
1.1 · Execution

Probe hybridization a–i

a

Set a thermomixer with heated lid to 42°C, or program a thermal cycler and start the program.

42°C Lid temperature
50 µl Instrument reaction volume
Overnight Run time
42°C · hold Pre-equilibrate
42°C Probe hybridization
16–24 h Hybridization duration
b

At room temperature, combine warm Hyb Buffer B and warm Enhancer in the order shown in the Hyb Mix table. Pipette-mix 10 times.

Room temperature state 10× pipette mix

Both components must remain warm and visibly free of precipitate. Do not place the prepared warm mix on ice.

c

Incubate the prepared Hyb Mix at 42°C for 5 min.

42°C temperature 5 min time
05:00
d

Centrifuge the fixed sample in Quenching Buffer B at 850 × g for 5 min at 4°C.

850 × g relative force 5 min time 4°C temperature
05:00
e

Remove the supernatant without disturbing the pellet.

For fewer than 300,000 fixed cells, use a swinging-bucket rotor and prioritize pellet retention. Complete supernatant removal is not required.

Up to 15 µl may remain. If needed, place 15 µl water or PBS in an empty tube of the same size as a visual reference.

f

Resuspend each pellet in 40 µl Hyb Mix.

40 µl Hyb Mix Room temperature sample state

For thermal-cycler incubation, the sample may be transferred to a tube strip. A 1.5-ml tube requires a thermomixer with heated lid; that format is preferred at ≤50,000 cells to avoid transfer-associated loss.

Keep the resuspended sample at room temperature. Do not place it on ice.

g

Add 10 µl of one unique Human or Mouse WTA Probe Barcode BC001–BC016 to the 40 µl sample/Hyb Mix suspension.

10 µl WTA probe 1 per tube assignment

Record the WTA Probe Barcode name and part number for every sample. Add only one WTA Probe Barcode to each sample tube.

h

Add 2 µl of one Antibody Multiplexing Barcode AB001–AB016 to each tube. Gently pipette-mix 10 times with the pipette set to 40 µl.

2 µl AB barcode 40 µl pipette setting 10× gentle mix

Record the Antibody Multiplexing Barcode name and part number for every sample. Add only one Antibody Multiplexing Barcode to each sample tube.

i

Incubate at 42°C for 16–24 h in a thermal cycler or in a thermomixer with heated lid and no shaking.

42°C temperature 16–24 h window No shaking mixing state

Do not shorten the incubation below 16 h.

Keep the incubation duration identical for all samples in the same experiment.

16:00:00 The allowed endpoint window closes 8 h after this timer reaches zero.
Local run record

Record assignments before incubation

These fields are a custom execution aid, not manufacturer-provided metadata. Entries are stored only in this browser’s local storage and are included when printing.

Sample and barcode run record
Sample ID Fixed cells / nuclei Species WTA barcode WTA PN AB barcode AB PN Notes
Protocol boundary

Stop after overnight hybridization.

The samples are now ready for the guide’s Step 2, Post-Hybridization Washing & Pooling. That procedure, its workflow choice, and its storage options are intentionally not included in this Step 1 page.