Apr 03, 2025
HyDrop v2 Bead Generation & Ligation Barcoding
- Suresh Poovathingal1,
- Marta Wojno1,
- Koen Theunis2,
- Florian De Rop2,3,4,5,
- Stein Aerts2,3,4,5
- 1VIB-KU Leuven Center for Brain & Disease Research, CBD Technologies, Single Cell & Microfluidics Expertise Unit, 3000 Leuven, Belgium;
- 2Laboratory of Computational Biology, VIB Center for AI & Computational Biology, Leuven, Belgium;
- 3VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium;
- 4Department of Human Genetics, KU Leuven, Leuven, Belgium;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, United States
- Aertslab
Protocol Citation: Suresh Poovathingal, Marta Wojno, Koen Theunis, Florian De Rop, Stein Aerts 2025. HyDrop v2 Bead Generation & Ligation Barcoding. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5n11dv1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 27, 2025
Last Modified: April 03, 2025
Protocol Integer ID: 125629
Keywords: ASAPCRN, hydrogel, microfluidics, hydrop, HyDrop, scATAC, scRNA, scOmics
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-025179
Aligning Science Across Parkinson’s
Grant ID: ASAP-000430
Abstract
This protocol details hydrogel bead generation and barcoding for HyDrop v2. The barcodes are build by split-pool and ligation strategy (3x96). Here, we will create an emulsion of acrylamide monomers in a carrier oil containing TEMED. The monomer droplets will polymerise and form hydrogel beads. Ideally, you have a bead stock of around 3 mL of beads before you barcode, but 2 mL can work as well. It is best to produce the beads in one single run, from a single bead mixture to prevent disparities in sizes and/or primer concentration within the bead from occurring.
Attachments
Materials
- 96-well plate
- Cassette HyDrop-v2-ATAC-plate
- Tris
- Tris-HCl
- NaCl
- NaOH
- MgCl2
- Tween-20
- dH2O
- Oligo-Mix BC1
- ATP
- T4 ligase
- Oligo-Mix B
- Brij-35
- TX-100
Before start
On the days before the barcoding prepare the primer cassette for the stage 1 and stage 2 barcoding.
Hydrogel bead generation
Hydrogel bead generation
Prepare monomer mix. The volume used here will approximately equate the final volume of dense bead stock that you will produce. The beads will be slightly larger than the droplets generated, but you will also generate small losses during the wash steps.
| A | B | C | D | E | |
| Vol (uL) | Stock | Final | Unit | ||
| TBSET | 110 | 1 | 0.1 | X | |
| Acrylamide | 269.5 | 40 | 10 | %w/v | |
| N,N'-Bis(acryloyl)cystamine | 45.83 | 4.8 | 0.2 | %w/v | |
| Ammonium persulfate | 66 | 10 | 0.6 | % | |
| Acrydite primer bead handle | 44 | 100 | 4 | uM | |
| Nuclease free water | 564.67 | ||||
| TOTAL | 1100 |
The volumes used here equals to 10% acrylamide (%T) and 2% bisacryloylcystoylamine (%C).
Note that N,N′-Bis(acryloyl)cystamine is dissolved w/v in methanol.
Add 1000 μL of filtered HFE-7500 Novac oil to the syringe (3 mL) and add the monomer mix and layer it on top of the HFE and prepare the syringe for injection.
Mix 2900 μL of the EA008 surfactant with 14.5 μL of TEMED (=0.5% TEMED). Mix the content well.
Prepare two 1.5 mL Eppendorf collection tubes and put 200 μL of mineral oil in the tubes. This will also serve as protection against evaporation during the overnight incubation afterwards.
Run the droplet generation for the preparation of the hydrogel beads.
Flow rates:
| A | B | C | |
| start | stable | ||
| monomer mix | 400 | 400 | |
| oil | 400 | 750 |
Collect the emulsion for 55 minutes in two 1.5 mL Eppendorf tubes.
Remove excess oil from the bottom of the collection tubes and double check if the layer of mineral oil is on top of the emulsions.
Put the emulsions on a 65°C heat block for 14 hours. Leave beads at 12°C until they are washed.
Remove top layer of mineral oil and bottom layer of EA008.
Wash the beads:
- 3 times with 20% PFO in HFE-7500.
- 3 times with 1% SPAN80-Hexane.
- 3 times with with TBSET buffer.
- 3 times with TET buffer and combine all aliquots in the last wash.
Filter the beads through a 70 um filter to exclude contamination of large beads.
Store the beads at 4°C.
Oligo preparation (done at least a day prior to barcoding)
Oligo preparation (done at least a day prior to barcoding)
Take 3 fresh 96 well plates. And label as:
- Cassette 1-HyDrop-v2-ATAC-plate (200 μM)
- Cassette 2-HyDrop-v2-ATAC-plate (200 μM)
- Plate 3-HyDrop-v2-ATAC-plate (500 μM)
For Cassette 1-HyDrop-v2-ATAC-plate:
Mix 20 µL of primers from plate-1-Fwd-96 of the 400 micromolar (µM) stock primer plate to 20 µL of primers from plate-1-Rev-96 of the 400 micromolar (µM) stock primer.
For Cassette 2-HyDrop-v2-ATAC-plate:
Mix 20 µL of primers from plate-2-Fwd-96 of the 400 micromolar (µM) stock primer plate to 20 µL of primers from plate-2-Rev-96 of the 400 micromolar (µM) stock primer.
For Plate 3-HyDrop-v2-ATAC-plate:
Aliquot 18 µL of primers from plate-3-ATAC-96 of the 500 micromolar (µM) stock primer plate to a new plate.
Barcoding Round 1
Barcoding Round 1
3h 38m
3h 38m
- Spin down the beads stored in TET buffer.
- Remove supernatant and wash the beads one more time with TET buffer.
- Centrifuge at 1000 x g, 00:03:00 , brake 8, and discard the supernatant.
3m
Prepare the pre-Ligation buffer (PL buffer) below and filter with 0.2 µm filter (if not available).
| A | B | C | D | E | F | |
| Component | Stock | Unit | Final | Unit | Volume | |
| Tris 8.0 | 1000 | mM | 10 | mM | 5000 | |
| NaCl | 5000 | mM | 30 | mM | 3000 | |
| MgCl2 | 1000 | mM | 1 | mM | 500 | |
| Tween-20 | 10 | % | 0.1 | % | 5000 | |
| dH2O | 486500 | |||||
| TOTAL | 500000 |
- Wash the beads with PL buffer 2X times.
- Centrifuge @ 1000 x g, 00:01:00 , brake 8, discard the supernatant.
1m
Using Hamilton, aliquot 22 µL of the compacted beads to each of the wells of 96 well plate.
Total compacted bead volume: 22×1.2×96 = 2534.4 µL .
Prepare the 2X Ligation-primer buffer (T4) below:
| A | B | C | D | E | F | G | |
| Component | Stock | Unit | Final | Unit | Volume | 96x1.2 | |
| Tris 7.5 | 1000 | mM | 100 | mM | 2 | 230.4 | |
| MgCl2 | 1000 | mM | 20 | mM | 0.4 | 46.08 | |
| Cassette 1-HyDrop-v2-ATAC | 200 | µM | 40 | µM | 4 | NO ADD | |
| dH2O | 13.6 | 1566.7 | |||||
| 20 | 16 µl per reaction | ||||||
| 1843.2 µl |
µl per reaction
Using Hamilton, aliquot 16 µL of the 2X ligation-primer buffer to the PCR plate.
- Add 4 µL (Hamilton) of Cassette 1-HyDrop-v2-ATAC-plate 200 micromolar (µM) , spin and mix well by vortexing and place on PCR block pre-heated to 75 °C (with heated lid to 105 °C ).
Heat the thermoblock to 75 °C and keep it ready.
- Heat the mix at 75 °C for 00:03:00 on the PCR and transfer and switch off the heat block to cool to Room temperature , to anneal the oligo to bead (~2.5 hours).
- Every 30 mins vortex the plate at max speed (2000x RPM) for 00:00:30 to resuspend the beads. Keep on looking after switching off for 02:30:00 (reaches 33 °C ).
- After 02:00:00 , leave the plate at Room temperature .
Note
Take it from block and keep it at Room temperature for 00:10:00 .
2h 33m 30s
In the meantime, prepare the 1X ligase mix below (T4):
| A | B | C | D | E | F | G | |
| Component | Stock | Unit | Final | Unit | Volume | 96×1.05 | |
| Tris 7.5 | 1000 | mM | 50 | mM | 0.5 | 52.8 | |
| MgCl2 | 1000 | mM | 10 | mM | 0.1 | 10.6 | |
| ATP | 10 | mM | 1 | mM | 5.1 | 538.6 | |
| T4 ligase | 400 | U/µl | 20 | U/µl | 2.5 | 252 | |
| dH2O | 1.8 | 181.4 | |||||
| 10 | 10 µl per reaction | ||||||
| 1035.4 µl |
- Using Mantis, aliquot 10 µL of the ligase mix to the 96 well plate, after sealing the plate vortex the content in a vortex shaker, centrifuge at 1000 x g, 00:00:30 .
- On thermoblock shake 2000 rpm, 00:00:30 , to mix the beads.
30s
Perform ligation at 25 °C for 01:00:00 with a shaking of 1000 rpm (every minute shake at 1600 rpm, 00:00:30 ).
1h
Perform Overnight incubation at 16 °C on a thermoblock with shaking of 1000 rpm, 00:00:30 . <Pause point - overnight>
Barcoding Round 2
Barcoding Round 2
3h 59m 30s
3h 59m 30s
Remove the plate from 16 °C incubation and leave at 4 °C till the next processing step (at least 30 min).
Prepare 2X ligation-primer buffer.
| A | B | C | D | E | F | G | |
| Component | Stock | Unit | Final | Unit | Volume | 96x1.2 | |
| Tris 7.5 | 1000 | mM | 100 | mM | 2 | 230.4 | |
| MgCl2 | 1000 | mM | 20 | mM | 0.4 | 46.1 | |
| dH2O | 13.6 | 1566.7 | |||||
| 20 | 16 µl per reaction | ||||||
| 1843.2 µl |
Aliquot 16 µL of the above 2X ligation-primer buffer mix to a fresh 96 well plate.
- Add 4 µL of Cassette 2-HyDrop-v2-ATAC-plate 200 micromolar (µM) .
- Mix well (vortex) and place on a PCR block pre-heated to 75 °C (lid 105 °C ).
- Heat the plates at 75 °C for 00:03:00 , transfer the plate to thermoblock at 75 °C and switch off the heat block to cool to Room temperature , to anneal the oligos.
- Keep on looking after switching off for 02:30:00 .
- After 2.5 hours, leave the plate at Room temperature .
Note
Take it from block and keep it at Room temperature for 00:10:00 .
2h 33m
- In the meantime, perform the STOP-25 cleanup on the Hamilton.
- Transfer to a 50 ml falcon.
- Incubate for 00:20:00 after collection.
20m
Spin the falcon at 3220 x g, 00:03:00 s and braking of 7. Transfer the beads to 15 ml falcon.
3m
Wash the beads with Stop-10 buffer. Centrifuge @ 1000 x g, 00:01:00 and discard the supernatant.
1m
Wash 3X times with TET buffer. Centrifuge @ 1000 x g, 00:01:00 and discard the supernatant.
1m
Wash the beads with PL buffer 2X times. Centrifuge @ 1000 x g, 00:01:00 and discard the supernatant. Set the level of beads to 2534.4 µL .
1m
Using Hamilton, aliquot 22 µL of the compacted beads to each of the wells of 96 well plate of a new deep well 96 well plate.
After the completion of the oligo annealing, spin the plate to collect condensate and using Hamilton transfer the contents of the annealed oligo cassette plate to the bead plate.
Prepare the ligase mix below:
| A | B | C | D | E | F | G | |
| Component | Stock | Unit | Final | Unit | Volume | 96×1.1 | |
| Tris 7.5 | 1000 | mM | 50 | mM | 0.5 | 52.8 | |
| MgCl2 | 1000 | mM | 10 | mM | 0.1 | 10.6 | |
| ATP | 10 | mM | 1 | mM | 5.1 | 538.6 | |
| T4 ligase | 400 | U/µl | 20 | U/µl | 2.5 | 252 | |
| dH2O | 1.8 | 181.4 | |||||
| 10 | 10 µl | ||||||
| 1035.4 |
Using Mantis, aliquot 10 µL of the ligase mix to the 96 well plate, after sealing the plate vortex the content in a vortex shaker, centrifuge 1000 x g, 00:00:30 . On thermoblock shake 1600 rpm, 00:00:30 , to mix the beads.
30s
Perform ligation at 25 °C for 01:00:00 with 1000 rpm rotation shaking (every minute shake at 1000 rpm, 00:00:30 ).
1h
Perform Overnight incubation at 16 °C on a thermoblock with 1000 rpm rotation shaking. <Pause point - overnight>
Barcoding Round 3
Barcoding Round 3
1h 32m 30s
1h 32m 30s
Remove the plate from 16 °C incubation and leave at 4 °C till ready for subsequent processing step.
Perform the STOP-25 cleanup on the Hamilton. Transfer to a 50 ml falcon. Incubate for 00:20:00 after collection.
20m
Spin the falcon at 3220 x g, 00:03:00 and braking of 7. Transfer the beads to 15 ml falcon.
3m
Wash the beads with Stop-10 buffer. Centrifuge @ 1000 x g, 00:03:00 and discard the supernatant.
3m
Wash 3X times with TET buffer. Centrifuge @ 1000 x g, 00:03:00 and discard the supernatant.
3m
Wash the beads with PL buffer 2X times. Centrifuge @ 1000 x g, 00:03:00 and discard the supernatant. Set the level of beads to 2534.4 µL .
3m
Using Hamilton, aliquot 23 µL of the compacted beads to a fresh 96 deep well plate.
Prepare the 2X Ligation buffer below:
| A | B | C | D | E | F | G | |
| Component | Stock | Unit | Final | Unit | Volume | 96x1.3 | |
| Tris 7.5 | 1000 | mM | 100 | mM | 2 | 249.6 | |
| MgCl2 | 1000 | mM | 20 | mM | 0.4 | 49.92 | |
| plate 3-HyDrop-v2-ATAC-plate | 500 | µM | 30 | µM | 3 | NO ADD | |
| dH2O | 14.6 | 1822.1 | |||||
| 20 | 17 |
Using Hamilton, aliquot 17 µL of the 2X ligation-primer buffer to the PCR plate.
Add 3 µL of plate 3-HyDrop-v2-ATAC-plate 500 micromolar (µM) .
Prepare the ligase mix below:
| A | B | C | D | E | F | G | |
| Component | Stock | Unit | Final | Unit | Volume | 96×1.1 | |
| Tris 7.5 | 1000 | mM | 50 | mM | 0.5 | 52.8 | |
| MgCl2 | 1000 | mM | 10 | mM | 0.1 | 10.6 | |
| ATP | 10 | mM | 1 | mM | 5.1 | 538.6 | |
| T4 ligase | 400 | U/µl | 20 | U/µl | 2.5 | 252 | |
| dH2O | 1.8 | 181.4 | |||||
| 10 | 10 µl | ||||||
| 1035.4 |
Using Mantis, aliquot 10 µL of the ligase mix to the 96 well plate, after sealing the plate vortex the content in a vortex shaker, centrifuge 1000 x g, 00:00:30 . On thermoblock shake 2000 rpm, 00:00:30 , to mix the beads.
30s
Perform ligation at 25 °C for 01:00:00 with rotation mixing of 1000 rpm .
Note
Every minute shake at 1600 rpm, 00:00:30 .
1h
Perform Overnight incubation at 16 °C on thermoblock with rotation mixing of 1000 rpm . <Pause point - overnight>
Bead Cleanup
Bead Cleanup
29m
29m
Remove the plate from 16 °C incubation and leave at 4 °C till ready for subsequent processing step.
Perform the STOP-25 cleanup on the Hamilton. Transfer to a 50 ml falcon. Incubate for 00:20:00 after collection.
20m
Spin the falcon at 3220 x g, 00:03:00 and braking of 7. Transfer the beads to 15 ml falcon.
3m
Wash the beads with Stop-10 buffer. Centrifuge @ 1000 x g, 00:03:00 and discard the supernatant.
3m
Wash 3X times with TET buffer. Centrifuge @ 1000 x g, 00:03:00 and discard the supernatant.
3m
Denaturation and Final Steps
Denaturation and Final Steps
24m
24m
Prepare the following denaturation buffer and filter with 0.2 µm filter.
| A | B | C | D | |
| Component | Stock | Final | Volume | |
| Water | - | - | 60.5 ml | |
| NaOH | 10 N | 938 µl | ||
| Brij-35 | 1050 µl | |||
| 62.5 |
Add 15 mL of denaturation solution to beads and incubate for 00:10:00 on a rotator at Room temperature .
10m
Wash the denaturation buffer 3X times with denaturation solution with 15 mL buffer.
Add 15 mL of Neutralization solution to beads and incubate for 00:10:00 on a rotator at Room temperature .
10m
Wash the beads with Neutralization buffer 1X more time.
Wash 3X times with TET buffer and proceed to QC.
Filter using 70 µm filter.
Prepare the following.
Wash 3X 15 mL falcon 3 times with distilled water to clear of the dust in the tube and keep (3X 15 ml Falcon) 2X Falcon is for TET buffer; 1X for Bead collection.
Wash 4X 50 mL falcon 3X times with distilled water to clear of the dust in the tube and keep (4X 50 ml Falcon) 2X Falcon is for bead filtering; 1X for Bead refiltering and 1X for filtered lysis buffer collection.
Sequential transfer of beads to 70 µm filter for 2 stage filtration.
Collect the final filtrate and transfer to the dust clean 15 mL falcon. Keep On ice .
Prepare the 1X lysis mix in fresh 50 ml falcon.
Prepare the 1X Lysis mix
| A | B | C | D | E | F | |
| Stock | Unit | Final drop | Final | Volume | ||
| Tris-HCl (pH 7) | 1000 | mM | 25 | 125 | 5000 | |
| NaCl | 5000 | mM | 30 | 150 | 1200 | |
| MgCl2 | 1000 | mM | 2.5 | 12.5 | 500 | |
| TX-100 | 100 | % | 0.25 | 1.25 | 500 | |
| BSA | 30 | % | 0.08 | 0.4 | 533 | |
| dH2O | - | 32800 | ||||
| 40000 |
Filter the 1X lysis mix to a 2nd pre-clean 50 ml falcon using 0.2 µm disc filter.
Spin down the falcon with beads in TET at 800 x g, 00:01:00 , remove as much as supernatant as possible.
1m
Add 10 mL of filter 1X lysis mix. Vortex well. Spin down the falcon with beads in TET at 1000 x g, 00:01:00 . Remove and discard the supernatant.
1m
Add 10 mL of filter 1X lysis mix. Vortex well. Incubate the beads at 4 °C Overnight for equilibration.
Next day wash the 8-strip Eppendorf tubes 2X times with the DI water. Remove as much liquid as possible with the p200 multichannel.
Spin down the bead at 1000 x g, 00:02:00 and aliquot 40 µL beads to 8-strip tube.
Put the strips to -80 °C storage.
2m