Oligonucleotide-polymer conjugation for imaging mass cytometry

Resuspend lyophilized oligos in ddH20 (RNAase and DNAse free water) to a final concentration of 250 µM in 1.5 mL eppendorf tubes

Determine concentration at NanoDrop.

Note: If there is a big discrepancy between what you measured and what should be there then rather use concentrations from your measurements and continue with those.

Oligonucleotide thiol reduction

Note: for the conjugation with the maleimide-polymers 2 nmol of oligos are needed. However, there is a purification step before so the starting material should be more. As little as 5 nmol starting material can work but it is recommended to start with 12.5-25 nmol and potentially use less when more experienced with the protocol.

Mix 50 µl of the 250 µM oligonucleotides with 5 µl or 0.5 M TCEP (final TCEP conc. 50 mM).

Mix by pipetting and incubate at room temperature for 30 min.

Move to the next step while incubating.

Note: This part comes from the Fluidigm protocol "Lanthanide Labeling of antibodies – PRD002 Version 11"

Spin the polymer tube for 10 seconds in a microfuge to ensure that the reagent is at the bottom of the tube.

Resuspend the polymer with 95 µL of L-Buffer and mix by pipetting

Add 5 µL of lanthanide metal solution to the tube (final concentration: 2.5 mM in 100 µL) and mix by pipetting

Incubate at 37 °C for 30−40 minutes in a water bath or heat block.

Add 5.5 µl volume of 3M NaAc (final conc 0.3 M) to the oligos and briefly vortex.

Add 150 µl (2.5 volumes) of 100% EtOH (-20°C) and vortex. and put it to -20°C for at least 30 min.

Incubate mix at -20°C for at least 30 min or longer.

Cool down an Eppendorf centrifuge 5430R (or similar) to 4°C

Add 100 µL of L-Buffer to a 3 kDa Amicon filter.

Add the 100 µL metal-loaded polymer mixture that incubated at RT to the filter containing the 200 µL L-Buffer.

Centrifuge at 12,000 x g for 25 minutes at RT in a table to centrifuge

Repeat the wash by adding 400 µL of C-Buffer to the filter and centrifuge at 12,000 x g for 45 minutes at RT.

Move to oligonucleotide purification.

Remove oligos from -20°C. Spin down oligos for at least 30 min at max speed (~30’000 rcf).

Note: Keep in mind the orientation of the tubes to find pellet which will be very small.

Remove tube from centrifuge and remove supernatant with a suction device or pipette carefully without disturbing the pellet.

Add 500 µl ice-cold 70% EtOH. Do not disturb the pellet and insert tubes in the centrifuge in same direction as before and spin for 3 min at max speed.

Remove tubes from centrifuge and remove supernatant with a suction device or pipette carefully without disturbing the pellet. Place tube in a rack on the bench, open the lid and briefly air-dry the pellet (1-3 min).

Resuspend pellet in 50 µl C-buffer.

Determine concentration at Nanodrop (ssDNA mode). Calculate the volume containing 2 nmol of the reduced oligo.

Remove the polymers from the table top centrifuge. The volume should be 20-30 µl.

Mix 2 nmol of the reduced oligo with the 20-30 µl of the polymer in C-buffer. Fill to 200 µl with C-buffer and mix with pipette.

Incubate for 2 h at RT.

After 2 hours add TCEP to the reaction to a final concetration of 5 mM (1:100 from 0.5 M stock). Incubate another 30 min at RT.

Transfer the reaction onto flat filter tube Microcon 30 and add 300 µl ddH₂O. Spin 12 min at 12’000 rcf.

Discard flow through and repeat for a total of 3 washes with 250µl DEPC water each (each 10 min).

After the final wahsing step the oligos are retained in the tiny residual volume on top of the filter! Resuspend oligos from the top of the filter by adding 50µl of ddH₂O water and transfer to new tube. Repeat with another 50 µl of ddH₂O water.

Determine concentration at Nanodrop (ssDNA).

Prepare a 4% agarose gel with TAE buffer with gelred (1x) or any other means available for nucleic acid detection.

Load the conjugated and unconjugated oligos (roughly 500 ng is well visible and gives you a good impression of your labeling efficiency) next to each other on the gel and run at 120 volts for

Note: Be careful, oligos are very small (~20 nucleotides) and run fast. No ladder required since the unconjugated oligos serve as a control.

Prepare a 10 or 1 µM mix of the labelled oligos with ddH₂O.

Store the oligos at 4°C for further use with e.g. RNAscope.

Note: The oligos are stable at 4°C for several months. However, one may also freeze an aliquot of the oligos for later usage. We have tried this in the past and thawed, conjugated oligos worked well. Avoid freeze-thaw cycles. For higher plexicity it is recommended to store the oligos at higher concentrations to avoid adding too much "water" to the hybridization reaction.