Preparation of dissolvable gel beads containing Oligo DNA with Microdroplet/Microsphere Generator
Experimental Purpose:In this application note, based on the barcode gel bead preparation method of 10X Genomics, the Microdroplet/Microsphere Generator is used to prepare highly monodisperse dissolvable polyacrylamide gel beads containing Oligo DNA (CV<5%). An acrylamide mixture solution (acrylamide, N, N’- bis (acryloyl) cystamine and primer solution) is used as the dispersed phase, and Drop-Surf Generation Oil (containing N, N, N’, N’- tetramethylethylenediamine) is used as the continuous phase. The cross-linking rate of Oligo DNA concentration is up to 40%.
As the basic unit of biological structure and function, cells differ greatly in morphological types. Combined with high-throughput sequencing, droplet-based single cell sequencing technologies have been successfully applied to analyzing RNA [1-3], DNA [4,5] and protein [6,7] in single cells. To track a single cell, it is necessary to encapsulate the gel beads containing DNA barcode together with the single cell in nanoliter-sized microdroplets for subsequent single cell analysis and sequencing [8]. The key to realize this technique is to add the synthesized DNA barcode to the gel beads.
Currently, there are several reported methods for preparing DNA barcode, including polyacrylamide gel beads used in inDrop[2], hydroxylated methacrylic acid polymer gel beads used in Drop-seq[3] and polyacrylamide gel beads used in 10X Genomics[8]. However, the gel beads produced by these methods has their own advantages and shortcomings. In the inDrop system, the gel beads can be closely packed in a microfluidic device channel to achieve more than 95% loading of single bead per drop. However, the release of DNA barcode in the process requires ultraviolet rays, which increases the difficulty of primer release[2]. Also, ultraviolet rays may also cause irreversible damage to DNA or RNA. In the Drop-seq system, the primers cannot be released from the beads, which means the reaction only take place on the surface of the beads and reduces the reaction efficiency. In 10X Genomics system, the beads can be effectively encapsulated and released, but the relatively high cost and the lack of flexibility in primer design on beads limit the development of some experiments. What thus missing today, is barcode beads that are easy to fabricate and efficiently deliver primers into drops, so as to achieve high detection efficiency.
Based on Wang et al.’s research [9] and the preparation method of barcode gel beads of 10X Genomics, FluidicLab provides an effective strategy to produce dissolvable polyacrylamide gel beads containing Oligo DNA. The schematic diagram is shown in the follow picture. The dispersed phase (containing acrylamide, ammonium persulfate, N, N’- bis (acryloyl) cystamine and primer solution), and the continuous phase (Drop-Surf Droplet Generation Oil and N, N, N’, N’- tetramethylethylenediamine) are pushed into a microfluidic chip by FluidicLab Microdroplet Generator, and droplets are generated and collected to obtain highly monodisperse dissolvable polyacrylamide gel beads containing Oligo DNA. The gel beads can be completely dissolvable by opening its disulfide bond with dithiol, resulting in releasing DNA barcode.
Reagents
Droplet Generation Oil (Drop-Surf, 2% surfactant by weight)
Demulsifier (Drop-Surf)
TBSET buffer (FluidicLab)
40% acrylamide solution (wt%, FluidicLab)
0.8% N, N’-bis (acryloyl) cystamine solution (wt%, BAC, FluidicLab)
Ammonium persulfate (APS, FluidicLab)
N, N, N’, N’-tetramethylethylenediamine (TEMED, FluidicLab)
For a visualized guide of this section, refer to Application Video: Preparation of dissolvable gel beads containing Oligo DNA with Microdroplet/Microsphere Generator.
1.Solution preparation:
(1)Preparation of 10% (wt%) ammonium persulfate (APS) solution: 0.1 g of APS is dissolved into pure water. Fix the volume to 10 mL. (2) Preparation of dispersed phase containing Oligo DNA ① 200 μL of TBSET buffer, 300 μL of 40% (wt%) acrylamide solution, 980 μL of 0.8% (wt%) N, N’-bis (acryloyl) cystamine solution and 120 μL of 10% (w/v) ammonium persulfate solution are mixed and filtered with a 0.22 μm syringe tip filter; ② 800 µL of acrylamide mixed solution, 166 µL of Oligo DNA with a concentration of 150 µM and 34 µL of pure water are mixed homogenously to obtain a 1 mL of dispersed phase solution containing Oligo DNA. The final composition is shown in the following table:
Item/unit
Initial concentration
Volume (μL)
Final concentration
Acrylamide mixed solution
TBSET buffer
/
100
/
Acrylamide solution (wt%)
40%
150
6%
N, N’- Bis (acryloyl) cystamine solution (wt%)
0.80%
490
0.392%
Ammonium persulfate (w/v)
10%
60
0.60%
Oligo DNA
150 μM
166
25 μM
Pure water
/
34
/
(3) Detection of Oligo DNA concentration in the dispersed phase: ① 5 µL of the dispersed solution containing Oligo DNA prepared above and 45 µL of pure water are mixed homogeneously for dilution; ② 298.5 µL of Qubit ssDNA Buffer and 1.5 µL of Qubit ssDNA Reagent are mixed homogenously to obtain a Qubit ssDNA detection reagent. ③ 199 µL of Qubit ssDNA detection reagent prepared in step ② and 1 µL of aqueous diluent prepared in step ① are mixed homogenously, and let stand for 2 min. Detect its concentration by Invitrogen Qubit4 Fluorometer. (4) Preparation of continuous phase: 12 µL of TEMED and 3 mL of Drop-Surf Droplet Generation Oil (1000:4, v/v) are mixed homogenously.
2. Preparation and cross-linking of beads containing Oligo DNA:
(1) Microfluidic devices set-up
For a more detailed installation and connection of Microdroplet/Microsphere Generator, refer to the User Guide V.1.0 Of Microdroplet/Microsphere Generator, section 2. Installation and connection of Microdroplet/Microsphere Generator. The connection is as follows (steps③-⑦ are shown in the figure below):
① Connect the devices with PU tubing in the following order:
The air compressor–the air source processing device–the Microdroplet/Microsphere Generator
② Connect the device to the power supply and PC, respectively.
③ Connect A0 (the pressure channel 1 outlet) and A1 (the continuous phase reservoir), B0 (the pressure channel 2 outlet) and B1 (the dispersed phase reservoir) with PU tubing, respectively.
④ Connect A1 (the continuous phase reservoir) and A2 (the flow sensor channel 1), B1 (the dispersed phase reservoir) and B2 (the flow sensor channel 2) with PEEK tubing and 1/4-28 UNF, respectively.
⑤ Connect A2 (the flow sensor channel 1) and A3 (the continuous phase inlet of the PDMS chip), B2 (the flow sensor channel 2) and B3 (the dispersed phase inlet of the PDMS chip) with PEEK tubing and 1/4-28 UNF, respectively.
⑥ C is a combination of a PDMS standard chip and a chip holder, which is sealed by 6 silicone connector seals.
⑦ The PEEK tubing is inserted into the chip & chip holder outlet (D) for emulsion output.
(2) Installition of FluidicLabSuite software:
Refer to the section 3.1 Installation of FluidicLabSuite Software in the User Guide V.1.0 Of Microdroplet/Microsphere Generator;
(3) Preparation of microdroplets containing Oligo DNA:
① 5 mL of continuous phase and 1 mL of dispersed phase are added into the corresponding reservoirs, respectively:
Reservoir 1 (controlled by Pressure Channel 1): the continuous phase;
Reservoir 2 (controlled by Pressure Channel 2): the dispersed phase.
② For the use of the integrated camera and flow sensors, refer to the section 3.2 Equipment Addition of FluidicLabSuite Software in the User Guide V.1.0 Of Microdroplet/Microsphere Generator.
③ Turn on the air compressor and air source treatment device.
④ A centrifuge tube is placed at the emulsion outlet to collect the pre-waste liquid.
⑤ Set the pressure of pressure channel 1 and 2 by FluidicLabSuite control to exhaust the air in the PEEK tubing and the microchannels of chip.
⑥ After the PEEK tubing and the microchannels of chip are filled with liquid, switch the control mode from pressure control to flow rate control. The flow rates of channels 1 and 2 are set as 25 and 12 μL/min, respectively.
⑦ The smooth output of target flow rate can be quickly achieved through adjusting the feedback value.
⑧ After a few minutes, collect a drop of the emulsion onto a hydrophobic substrate base, and ensure the uniformity of droplet size with an optical microscope.
⑨ After that, collect the emulsion into a 1.5 mL centrifuge tube containing cross-linking reagent solution.
⑩ Collect the emulsion for 20 minutes, add 200 μL of mineral oil on the top of the droplets, then seal the emulsion and heat to 65 ℃ overnight for cross-linking gelation.
3. Aftertreatment of beads containing Oligo DNA:
1) Remove the mineral oil (at the top of the container) and the oil phase (at the bottom of the container) with a pipette.
2) Add 2x volume of Drop-Surf Demulsifier to the beads. For every 100 μL beads, add 200 μL Drop-Surf Demulsifier.
3) Vortex the mixture for 20 seconds, then centrifuge it at 5000 rpm for 30 seconds. Remove Drop-Surf Demulsifier at the bottom of the container.
4) Repeat steps 2) and 3) 1~2 times, until all white beads on the top of the container change into transparent.
5) Add 2x volume of 1% Span 80 in n-hexane solution to the beads. For every 100 μL beads, add 200 μL 1% Span 80 in n-hexane solution.
6) Vortex the mixture for 20 seconds, then centrifuge it at 5000 rpm for 30 seconds. Remove the n-hexane at the upper of the container.
7) Repeat steps 5) and 6) 1~2 times.
8) Add 3x volume of TET buffer to the beads. For every 100 μL beads, add 300 μL TET buffer.
9) Vortex the mixture for 20 seconds, then centrifuge it at 5000 rpm for 5 minutes. Remove buffer at the upper of the container.
10) Repeat steps 8) and 9) 1~2 times.
11) Finally, disperse the beads in TET buffer. The prepared beads can be stored at 4 ℃ for up to 6 months.
4. Detection of Oligo DNA concentration in beads:
① 5 µL of the beads containing Oligo DNA prepared above and 45 µL of 10 mM dithiol (DTT) solution are mixed homogenously for complete dissolution;
② 298.5 µL of Qubit ssDNA Buffer and 1.5 µL of Qubit ssDNA Reagent are mixed homogenously to obtain a Qubit ssDNA detection reagent.
③ 199 µL of Qubit ssDNA detection reagent prepared in step ② and 1 µL dissolved solution prepared in step ① are homogenously mixed, and let stand for 2 min. Detect its concentration by Invitrogen Qubit4 Fluorometer.
5. Cleaning of microdroplets/microsphere Generator:
The PEEK Tubing, the flow sensors and the microchannels of the chip should be cleaned after the experiment. Otherwise, the reagents remaining in the flow channels could possibly damage the flow sensors and clog the microchannels of the chip. For details, please refer to the Instruction Card of Microdroplet/Microsphere Generator.
① The average particle size of the chitosan microdroplets is 45.16 μm, and have extremely high monodispersity (CV=2.65%). The picture and particle size distribution are shown as follow:
② After cross-linking gelation, the average particle size of bead is 53.64 μm, and have extremely high monodispersity (CV=3.88%). The picture and particle size distribution are shown as follow:
③The concentration of Oligo DNA in the aqueous phase is 14.9 μg/mL (24.35 μM) detected with Invitrogen Qubit4 Fluorometer.
④ The concentration of Oligo DNA in the beads is 6.48 μg/mL (10.58 μM) detected with Invitrogen Qubit4 Fluorometer.
Key points of the experiment
1.This application note is only suitable for the reagent kit provided by FluidicLab. Other reagents may not be applicable. 2.FluidicLab Standard PDMS Hydrophobic Chips are strongly recommended for your success preparation of beads using this specific application note. 3.Note that the sizes of the beads are larger than that of the microdroplets, thus the parameters (e.g. flow rates) should be carefully adjusted to obtain a desired result in diameters.
References
[1]Prakadan S. M., et al. Scaling by shrinking: empowering single-cell’omics’ with microfluidic devices, Nat. Rev. Genet., 18, 345 (2017).
[2] Klein A. M., et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells, Cell,161, 1187 (2015).
[3] Macosko E. Z., et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets, Cell,161, 1202 (2015).
[4] Lan F., et al. Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding, Nat. Biotechnol., 35, 640 (2017).
[5] Lareau C. A., et al. Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility, Nat. Biotechnol., 37, 916 (2019).
[6] Stoeckius M., et al. Simultaneous epitope and transcriptome measurement in single cells, Nat.Methods,14, 865 (2017).
[7] Peterson V. M., et al. Multiplexed quantification of proteins and transcripts in single cells, Nat.Biotechnol., 35, 936 (2017).
[8] Zheng G. X. Y., et al. Massively parallel digital transcriptional profiling of single cells, Nat. Commun., 8, 14049 (2017).
[9] Wang Y., et al. Dissolvable polyacrylamide beads for high-throughput droplet DNA barcoding, Adv. Sci., 7, 1903463 (2020).
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