Preparation of CITE-seq oligos and antibodies:
Biotinylation of oligos
o Order 5’amine-labelled oligos with a C12 spacer and a specific barcode:
• Small synthesis scales of ~25 nmoles are sufficient for many antibody conjugations.
o Clean oligo by Ethanol precipitation to remove residual synthesis contaminants:
• Resuspend all 25 nmoles of lyophilized oligo in 200 µl 0.5M NaCl.
• Centrifuge full speed (~18,000g) in table top centrifuge for 5 minutes at room temperature.
• Carefully transfer supernatant to new tube if there is visible pellet. Pellet is residual resin from oligonucleotide synthesis.
• Add 3 volumes (600 µl) of 100% EtOH.
• Incubate at -80˚C for at least 30 minutes.
• Centrifuge full speed (~18,000g) in table top centrifuge for 30 minutes at 4˚C.
• Wash pellet in 80% EtOH.
• Centrifuge full speed (~18,000g) in table top centrifuge for 30 minutes at 4˚C.
• Wash pellet in 80% EtOH.
• Centrifuge full speed (~18,000g) in table top centrifuge for 30 minutes at 4˚C.
• Air dry pellet.
o Resuspend oligo pellet in 20 µL PBS pH 8.5.
o Biotinylate oligo with EZ-link Sulpho-NHS S-S Biotin per manufacturer protocol (see materials):
• Resuspend Single Use EZ-link NHS S-S Biotin in 164 µl DMSO to make 10mM solution.
• Add 10 µl of Biotin-NHS to oligo (100 nmoles ~4 fold excess over oligo).
• Incubate at RT for 5 minutes.
• Repeat 4 more additions of 10 µl Biotin-NHS to oligo and incubations for 5 minutes. Final volume now 70 µl.
• Purify oligo by Ethanol precipitation:
• Increase volume to 400 µl (add 330 µl water) to reduce DMSO concentration to <10%.
• Add 1:10 vol. 5M NaCl (40 µl).
• Add 3 vol. 100% Ethanol (1.2 mL).
• Incubate for at least 30 minutes at -80˚C.
• Centrifuge full speed (~18,000g) in table top centrifuge for 30 minutes at 4˚C.
• Wash pellet in 500 µl 80% EtOH.
• Centrifuge full speed (~18,000g) in table top centrifuge for 30 minutes at 4˚C.
• Resuspend pelleted oligo in 20 µl PBS pH 8.5.
• Add 10 µl of Biotin-NHS to oligo (100 nmoles ~4 fold excess over oligo).
• Incubate at room temperature for 5 minutes.
• Repeat 4 more additions of 10 µl Biotin-NHS to oligo and incubations for 5 minutes. Final volume now 70 µl.
o Clean oligos vigorously to eliminate biotin carryover by two steps:
- Ethanol precipitation:
• Increase volume to 400 µl (add 330 µl water) to reduce DMSO concentration to <10%.
• Add 1:10 vol. 5M NaCl (40 µL).
• Add 3 vol. 100% Ethanol (1.2 mL).
• Incubate for at least 30 minutes at -80˚C.
• Centrifuge full speed (~18,000g) in table top centrifuge for 30 minutes at 4˚C.
• Wash pellet in 500 µl 80% EtOH.
• Centrifuge full speed (~18,000g) in table top centrifuge for 30 minutes at 4˚C.
• Wash pellet in 500 µl 80% EtOH.
• Centrifuge full speed in (~18,000g) table top centrifuge for 30 minutes at 4˚C.
• Air dry pellet shortly.
• Resuspend pellet in water at an estimated concentration of >100 µM ~200 µL.
- Size exclusion on Bio-Spin P6 desalting column according to manufacturer’s protocol:
• Spin oligos at 1000g for 4 minutes at room temperature.
• Collect flow through containing biotinylated purified oligo.
o Quantify oligo, if needed adjust concentration to 100 µM with TE and store at -20˚C.
o Verify sufficient biotinylation by running non-labelled control and biotinylated oligo on Agilent Bioanalyzer Small RNA Chip.
• If less than 90% of oligo is biotinylated, repeat biotinylation.
Streptavidin labelling of antibodies
• Only use flow cytometry optimized monoclonal antibody clones.
• Verify antibody concentration, 15 µg of antibody are needed for conjugation.
• Clean 15 µg of antibody on 50 kDa cutoff column per manufacturer protocol to exchange buffer and remove contaminants:
• Pre-wet 50 kDa cutoff column by adding 200 µl PBS pH 8.5.
• Combine 15 µl antibody with 200 µl PBS pH 8.5 and transfer to column.
• Spin at room temperature 4 minutes 14,000g.
• Discard flowthrough.
• Add 400 µl PBS pH 8.5 to column.
• Spin at room temperature 4 minutes at 14,000g until all liquid has drained to 20 µl mark on column.
• Recover concentrated purified antibody by placing column upside down in new tube and spin for 2 minutes at 3,000g.
• Adjust volume of recovered purified antibody to 30 µl with PBS pH 8.5.
• Streptavidin label antibodies using a 10 µg streptavidin kit (see materials) per manufacturer’s protocol with the following modifications:
• Note: 10µg streptavidin kit conjugates ~ 2 streptavidin tetramers to each antibody on average when using 15 µg of Antibody input.
• Add 3 µl of modifier solution (from kit) to 30 µl recovered purified antibody.
• Add purified antibody solution containing modifier directly onto the lyophilized reactive 10µg streptavidin.
• Mix by flicking the tube carefully.
• Incubate for at least 3 hours (or overnight) at room temperature.
• Quench reaction by adding 3 µl quenching solution (from kit).
• Add 4 µl 5M NaCl to increase the NaCl concentration to ~0.5M.
• Add 4 µl Tween 20 (0.1% in H2O) to get final of ~0.01% Tween.
• Antibodies are now ready to be attached to biotinylated oligos without additional cleanup steps (see below).
Merge streptavidin-antibodies with biotinylated-oligos in PBS/0.5M NaCl.
• Note: Each antibody should be labelled with 2 streptavidin molecules according to the kit specifications. 10µg streptavidin = 200pmol X 4 = 800pmol (biotin binding sites)
If all binding sites are saturated each antibody will have 8 oligos on average.
o Add ~800 pmoles of biotinylated purified oligo directly onto streptavidin antibody reaction tube.
o Incubate overnight at room temperature.
o Wash oligo-labelled antibodies on 50 kDa cutoff column per manufacturer’s protocol.
• Pre wet 50 kDa cutoff column with ~300 µl PBS.
• Transfer to oligo-labelled antibody to 50 kDa cutoff column.
• Spin at room temperature for 4 minutes at 14,000g.
• Discard flow through.
• Wash antibody-oligo-conjugate 7 times in 0.5M NaCl/PBS on column (Spin at RT 4min 14,000g, per cutoff column protocol).
• Perform the final wash with 1x PBS.
• Spin at room temperature 4 minutes at 14,000g until all liquid has drained to 20 µl mark on column.
• Recover concentrated purified antibody by placing column upside down in new tube and spin for 2 minutes at 3,000g.
• Adjust volume of recovered purified antibody to 30 µl with PBS.
o Validate oligo-conjugation by running ~0.7 µg of Antibody on 4% Agarose E-gel.
• Release oligo by treating with 0.2M DTT for 10min at 90˚C and compare to untreated antibody.
• Run ~0.7 µg of antibody complex on 4% Agarose gel (E-gel) for 4 minutes.
• Cool gel before visualization on ice.
o Store Antibodies in storage buffer at 4˚C until use (PBS, 1 µg/µl BSA, 0.05% Sodium Azide).
o Keep barcoded antibodies as pure stocks. Pool with other labeled antibodies only directly before use.
• Note that we have not extensively tested the shelf life of these conjugates. We recommend using the antibody-oligo complexes within a few weeks.
• If antibodies were not used for a prolonged period of time (> 3 months) it is advisable to run an aliquot on a 4% agarose gel (Figure 2) to verify oligos are still attached.
CITE-seq run:
Prepare antibody panel shortly before CITE-seq run
o Make antibody panel by pooling all antibodies and clean pooled panel on 50kDa cutoff column per manufacturer’s protocol to remove unbound oligos shortly before CITE-seq run:
• Use 1-2 µg of each antibody-oligo complex, comparable to what is recommended for flow cytometry.
• Optionally, optimal antibody concentration can be titrated by testing different concentrations.
• Merge appropriate amounts of all antibodies for one CITE-seq run in ~300 µl 0.5M NaCl/PBS containing 2 µl of 10 mM biotin to block unoccupied biotin-binding sites in streptavidin.
• Incubate for 5 minutes at room temperature.
• Pre-wet 50kDa cutoff column with ~100 µl 0.5M NaCl/PBS.
• Transfer biotin-blocked antibody panel to 50kDa cutoff column.
• Spin at RT 4 minutes 14,000g.
• Discard flow through.
• Wash antibody-panel 2 times in 400 µl 0.5M NaCl/PBS on column (Spin at RT 4min 14,000g, per cutoff column protocol).
• Perform the final wash with 400 µl 1x PBS.
• Spin at RT 4 minutes at 14,000g until liquid has drained to 20 µl mark on column.
• Recover concentrated purified antibody by placing column upside down in new tube and spin for 2 minutes at 3,000g.
• Adjust volume to 100 µl with cell staining buffer (2%BSA/0.02%Tween, PBS)
o Use pool for cell labelling immediately. Do not store merged antibody-oligo pool.
Cell staining for Drop-seq or 10X
o Carefully count cells to ensure accurate quantitation.
• Make note of cell viability (>95%) and also include dead cells in the total cell count!
• If you observe many dead cells live cell enrichment by FACS is recommended!
o Resuspend ~500,000 cells in 100 µl Staining buffer (2%BSA/0.02%Tween, PBS).
o Add 5 µl Fc Blocking reagent (FcX, BioLegend).
o Incubate 10 minutes at 4˚C.
o Add cleaned 100 µl Antibody-oligo pool (containing ~1-2 µg of each Antibody or titrated amounts).
• Final volume is now 200 µl.
o Incubate for 30 minutes at 4˚C.
o Wash cells 3 times with 1 mL Staining buffer (2%BSA/0.02%Tween, PBS), spin 5 minutes 450g at 4˚C.
o Resuspend cells in PBS at appropriate concentration for downstream application.
(e.g. for 10x [500 cells/µl] or Drop-seq [ 200 cells/µl]).
o Verify cell concentration by counting on hemocytometer.
Run Drop-seq or 10x Genomics single cell 3’ assay according to Macosko et al., 2015 (Drop-seq) or manufacturer’s instructions (10x Genomics) until after the cDNA amplification step.
After cDNA amplification: Separate ADTs (~180bp) and cDNAs (>300bp).
o Perform SPRI selection to separate ADTs and full length cDNAs.
o DO NOT DISCARD SUPERNATANT FROM 0.6X SPRI. THIS CONTAINS THE ADTs!
• Add 0.6X SPRI.
• Incubate 5 minutes and place on magnet.
• Supernatant contains ADTs.
• Beads contain full length cDNAs.
o cDNA >300bp (beads fraction).
• Proceed with standard 10x or Drop-seq protocol for cDNA sequencing library preparation.
o ADTs ~180bp (supernatant fraction).
• Purify ADTs using two 2XSPRI purifications per manufacturer protocol:
• Add 1.4X SPRI to supernatant to obtain a final SPRI volume of 2XSPRI.
• Incubate 10 minutes at room temperature.
• Place tube on magnet and wait ~2 minutes until solution is clear.
• Carefully remove and discard the supernatant.
• Add 400 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (only one Ethanol wash).
• Carefully remove and discard the ethanol wash.
• Centrifuge tube briefly and return it to magnet.
• Remove and discard any remaining ethanol.
• Resuspend in beads in 50 µl water.
• Perform another round of 2X SPRI purification by adding 100 µl SPRI reagent directly onto resuspended beads.
• Incubate 10 minutes at room temperature.
• Place tube on magnet and wait ~2 minutes until solution is clear.
• Carefully remove and discard the supernatant.
• Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (1st Ethanol wash).
• Carefully remove and discard the ethanol wash.
• Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (2nd Ethanol wash).
• Carefully remove and discard the ethanol wash.
• Centrifuge tube briefly and return it to magnet.
• Remove and discard any remaining ethanol and allow the beads to air dry for 2 minutes.
• Resuspend beads in 45 µl water.
• Pipette mix vigorously and incubate at room temperature for 5 minutes.
• Place tube on magnet and transfer clear supernatant to PCR tube.
• Amplify ADT sequencing library:
• Prepare 100uL PCR reaction with purified ADTs:
o 45 µl purified ADTs.
o 50 µl 2x KAPA Hifi PCR Master Mix.
o 2.5 µl Truseq Small RNA RPIx primer (containing i7 index) 10uM.
o 2.5 µl P5 oligo at 10uM depending on application:
♣ For Dropseq use P5-SMART-PCR hybrid oligo.
♣ For 10x use Illumina PE 1.0 P5 oligo.
o Cycling conditions:
95˚C 3 min
95˚C 20 sec |
60˚C 30 sec | 8-12 cycles
72˚C 20 sec |
72˚C 5 min
• Purify PCR product using 1.6X SPRI purification by adding 160 µl SPRI reagent.
• Incubate 5 minutes at room temperature.
• Place tube on magnet and wait 1 minute until solution is clear.
• Carefully remove and discard the supernatant.
• Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (first Ethanol wash).
• Carefully remove and discard the ethanol wash.
• Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (second Ethanol wash).
• Carefully remove and discard the ethanol wash.
• Centrifuge tube briefly and return it to magnet.
• Remove and discard any remaining ethanol and allow the beads to air dry for 2 minutes.
• Resuspend beads in 20 µl water.
• Pipette mix vigorously and incubate at room temperature for 5 minutes.
• Place tube on magnet and transfer clear supernatant to PCR tube.
• ADT library is now ready to be sequenced.
• Quantify library by standard methods (QuBit, BioAnalyzer, qPCR).
ADT library will be between 170-180 bp.