Jun 22, 2026
- Yifan Liu1,
- Yanan Du1
- 1School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China
- CAP-seq
Protocol Citation: Yifan Liu, Yanan Du 2026. CAP-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm8rp6g3p/v1
Manuscript citation:
Li L, Zhang R, Chen L, Tian X, Li T, Pu B, Ma C, Ji X, Ba F, Xiong C, Shi Y, Mi X, Li J, Keasling JD, Zhang J, Liu Y. Permeability-Engineered Compartmentalization Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems. Adv Sci (Weinh). 2022 Dec;9(34):e2203652. doi: 10.1002/advs.202203652.
Li J, Du Y, Shi Q, Chen L, Gao X, Liu Y, Luo Y. Permeability-Engineered Compartmentation System-Enabled Digital PCR (PECS-dPCR): A Digital Platform toward Multistep Biomolecular Assays. Anal Chem. 2024 Dec 31;96(52):20465-20474. doi: 10.1021/acs.analchem.4c04270.
Du Y, Wen C, Luo Y, Liu Y. Deterministic Co-encapsulation of Microparticles in Droplets via Synchronized Merging for Single-Cell Genomics. Anal Chem. 2025 Dec 2;97(47):26230-26236. doi: 10.1021/acs.analchem.5c05488.
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: May 06, 2026
Last Modified: June 22, 2026
Protocol Integer ID: 316484
Keywords: microbe genomics platform, thousands of singleamplified genome, singleamplified genome, seq microbial community, semipermeable encapsulation with minimal microfluidic, hydrogel, minimal microfluidic, sequencing depth
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Abstract
Microbial communities are extraordinarily diverse and play crucial roles in health and disease, yet current methods lack the resolution and scalability needed to dissect their genomic and ecological complexity at the single-cell level. Here, we present CAP-seq, a high-throughput single-microbe genomics platform that combines hydrogel-based semipermeable encapsulation with minimal microfluidics to recover thousands of singleamplified genomes (SAGs) with long reads and high completeness at low sequencing depth.
Protocol materials
SYBR GreenBiotiumCatalog #40086
PBSThermo Fisher
perfluorooctane (PFO)Merck MilliporeSigma (Sigma-Aldrich)
Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P6585
DPBSGibco - Thermo Fisher ScientificCatalog #C14190500BT
SDSThermo Fisher
EDTAThermo Fisher
Proteinase K Solution (20 mg/mL)Thermo FisherCatalog #AM2546
NaClThermo Fisher
Tag bufferABclonalCatalog #RK20239
Tag Enzyme 50ABclonalCatalog #RK20239
nuclease-free waterInvitrogen - Thermo Fisher
Phusion High-Fidelity DNA PolymeraseNEBCatalog #M0530L
Phusion High-Fidelity Reaction BufferNEBCatalog #M0530L
dNTPsVazymeCatalog #P031-01
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9779
Deep Vent DNA Polymerase NEBCatalog #M0258L
ET-SSBNEBCatalog #M2401S
10 X PCR stabilizerDP-BioCatalog #RRF0981
PEGDA, M.W. 8,000Alfa AesarCatalog #046801.03
PEGDA, M.W. 575Sigma aldrich.comCatalog #437441
Dextran 500KDaYeasen BiotechnologyCatalog #61216ES25
Lithium phenyl-2,4,6-trimethylbenzoylphosphinateMilliporeSigmaCatalog #900889
HFE75003M corporation
TE bufferThermofisher
NaOH
acetic acid
KAPA HiFi HotStart ReadyMixRocheCatalog #07958935001
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Native Barcoding Kit 24 V14 (SQK-NBD114.24)Oxford Nanopore TechnologiesCatalog #SQK-NBD114.24
EDTAThermo Fisher
Triton X-100Thermo FisherCatalog #85111
LysozymeLucigenCatalog #R1804M
Tris-8.0Thermo Fisher
lysostaphinMerck MilliporeSigma (Sigma-Aldrich)Catalog #L7386
NaClThermo Fisher
HEPESGibco - Thermo Fisher Scientific
Phi29 PolymeraseVazymeCatalog #N106-02
Pyrophosphatase, inorganic (0.1 U/µL)Thermo FisherCatalog #EF0221
10 X phi29 reaction bufferVazymeCatalog #N106-02
Pluronic F68Merck MilliporeSigma (Sigma-Aldrich)
plastic slidesThermo FisherCatalog #C10228
Microfluidics
Design the microfluidic devices using AutoCAD v2023 and Fabricate the devices by PDMS-based soft lithography.
Before use, treat the channels with Aquapel to render the channel surfaces hydrophobic.
Assemble the microfluidic station with an inverted microscope, high-speed camera, and six syringe pumps.
Load the required aqueous and oil phases into syringes and connect them to the corresponding device inlets.
Run each experiment using the specified inlet flow rates.
Generation of CAPs
34m
Prepare the aqueous two-phase system (ATPS) premix in a 1.5 mL centrifuge tube. For a 1 mL premix, include DPBSGibco - Thermo Fisher ScientificCatalog #C14190500BT , 0.055 g Dextran 500KDaYeasen BiotechnologyCatalog #61216ES25 , 0.03 g PEGDA, M.W. 8,000Alfa AesarCatalog #046801.03 30 µL PEGDA, M.W. 575Sigma aldrich.comCatalog #437441 and 100 µL of 4% (w/v) Lithium phenyl-2,4,6-trimethylbenzoylphosphinateMilliporeSigmaCatalog #900889 .
Mix the premix thoroughly and then centrifuge the tube at 14000 rpm, 00:30:00
Collect the upper PEGDA-rich phase and the lower dextran-rich phase separately for subsequent emulsification.
Generate CAPs using a flow-focusing microfluidic chip by emulsifying the ATPS in fluorinated oil containing 2% v/v PEG-PFPE surfactant.
Collect the CAPs in a tube. Irradiate under a 200 W UV lamp for 00:04:00 to gel the outer PEGDA-rich layer.
Characterization of CAPs
Characterize the semi-permeability of the CAPs using a customized PCR reaction inside the CAPs through amplifing RhaT gene fragments of different lengths from encapsulated E. coli genomic DNA to determine the dsDNA size threshold retained by CAPs.
Follow the detailed characterization procedures described in previous reports.
Citation
LINK
Citation
LINK
Stain CAPs containing intact cells, lysed cells, or amplified genomes with SYBR GreenBiotiumCatalog #40086 .
Examine the stained CAPs under a fluorescence microscope.
Cell preparation
Culture E. coli strains BL21 and MACH1, B. subtilis strain ster164, S. aureus strain RN4220, and K. pneumoniae strain KP_1.6366 separately in5 mL LB medium. Incubate at 37 °C Overnight in a shaking incubator.
Prepare serial dilutions of each overnight liquid culture. Load the diluted cell suspensions onto plastic slidesThermo FisherCatalog #C10228 .
Count the cells manually under a microscope and calculate the cell concentration based on the dilution factor and manual cell counts.
Cell encapsulation
Resuspend the cell samples in PBSThermo Fisher .
Add the cell suspension to the dextran-rich solution, which serves as the inner phase of the CAPs. Adjust the final cell density in the dextran-rich solution to 600 CFU/µL to achieve an expected CAP-positive rate of approximately 5%, according to Poisson statistics, to minimize doublet formation.
Generate CAPs using the microfluidic device. Collect the generated CAPs in a 1.5 mL centrifuge tube and gel the CAPs by UV irradiation.
Extract the gelled CAPs from the oil phase by adding an equal volume of 20 % volume perfluorooctane (PFO)Merck MilliporeSigma (Sigma-Aldrich) in fluorinated oil.
Wash the extracted CAPs three times with DPBS buffer containing 0.1 % volume Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P6585 .
Cell lysis
3h
Transfer 500 µL of dense-packed CAPs to a tube.
Prepare500 µL enzymatic lysis buffer containing the following components:
20 millimolar (mM) Tris-8.0Thermo Fisher
100 millimolar (mM) NaClThermo Fisher
10 millimolar (mM) EDTAThermo Fisher
1 % volume Triton X-100Thermo FisherCatalog #85111
1800 U LysozymeLucigenCatalog #R1804M
20 U lysostaphinMerck MilliporeSigma (Sigma-Aldrich)Catalog #L7386 .
Resuspend the CAPs with the enzymatic lysis buffer. Incubate at37 °C Overnight .
Wash the CAPs three times with PBS buffer containing10 millimolar (mM) EDTAThermo Fisher .
Prepare 500 µL of proteinase K lysis buffer containing the following components: 100 millimolar (mM) NaClThermo Fisher
10 millimolar (mM) EDTAThermo Fisher
1 % (v/v) SDSThermo Fisher
1 mg Proteinase K Solution (20 mg/mL)Thermo FisherCatalog #AM2546 .
Mix the washed CAPs with the proteinase K lysis buffer. Incubate at 55 °C
00:30:00 .
30m
Wash the CAPs three times with washing buffer containing the following components: 10 millimolar (mM) HEPESGibco - Thermo Fisher Scientific
2 % volume Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P6585
20 millimolar (mM) EDTAThermo Fisher
0.1 millimolar (mM) NaClThermo Fisher .
Resuspend the washed CAPs in DPBSGibco - Thermo Fisher ScientificCatalog #C14190500BT containing 0.1 % (v/v) Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P6585 .
Whole genome amplification
2h 10m
Transfer 300 µL of dense-packed CAPs after cell lysis to a tube.
Prepare 300 µL MDA mix containing the following components: 10 X phi29 reaction bufferVazymeCatalog #N106-02
30 µL Phi29 PolymeraseVazymeCatalog #N106-02
60 µL Pyrophosphatase, inorganic (0.1 U/µL)Thermo FisherCatalog #EF0221
2 millimolar (mM) dNTPsVazymeCatalog #P031-01
0.25 % volume Triton X-100Thermo FisherCatalog #85111
0.25 % volume Pluronic F68Merck MilliporeSigma (Sigma-Aldrich) .
Resuspend the CAPs with the MDA mix. Incubate at 30 °C for 02:00:00 to amplify genomic DNA inside the CAPs.
2h
Terminate the MDA reaction by heating the mixture at 65 °C for 00:10:00 .
10m
Wash the post-MDA CAPs three times with washing buffer.
Resuspend the washed post-MDA CAPs in DPBSGibco - Thermo Fisher ScientificCatalog #C14190500BT containing 0.1 % (v/v) Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P6585 .
Tagmentation
10m
Transfer 200 µL of post-MDA CAPs to a tube.
Prepare 200 μL tagmentation solution containing the following components:
80 µL Tag bufferABclonalCatalog #RK20239
40 µL Tag Enzyme 50ABclonalCatalog #RK20239
80 µL nuclease-free waterInvitrogen - Thermo Fisher
Resuspend the post-MDA CAPs with the tagmentation solution. Incubate at 55 °C 00:10:00 .
10m
Wash the tagmented CAPs three times with washing buffer.
Resuspend the washed CAPs in DPBS buffer containing 0.1% Tween-20.
Genome Barcoding
2h
Prepare the linear amplification mix for genome barcoding:
120 µL Phusion High-Fidelity Reaction BufferNEBCatalog #M0530L
16 µL Phusion High-Fidelity DNA PolymeraseNEBCatalog #M0530L
16 µL Deep Vent DNA Polymerase NEBCatalog #M0258L
12 µL ET-SSBNEBCatalog #M2401S
64 µL 100 millimolar (mM) DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9779
64 µL 10 millimolar (mM) dNTPsVazymeCatalog #P031-01
64 µL 10 X PCR stabilizerDP-BioCatalog #RRF0981
16 µL nuclease-free waterInvitrogen - Thermo Fisher .
Co-encapsulate the tagmented CAPs, Barcode beads (PercentBio, RRF0991) and linear amplification mix using a custom bead-pairing device.
Merge the droplets so that each resulting droplet contains one CAP, one dissolved barcode bead carrying a unique barcoding primer, and the linear amplification mix. The detailed procedure was performed as described in our previously published work.
Citation
LINK
18) Collect the droplets in PCR tubes.
Note
Replace the original emulsification oil with Bio-Rad QX200 oil before thermal cycling.
Perform linear amplification using the following thermal cycling program:
72 °C 00:05:00
98 °C 00:03:00
15 cycles of:
98 °C 00:00:10
60 °C 00:00:30
72 °C 00:05:00
Final incubation of:
4 °C Hold
All ramp times are at 1 °C per second.
2h
CAP dissolution
10m
After barcoding, add an equal volume of 20 % (v/v) perfluorooctane (PFO)Merck MilliporeSigma (Sigma-Aldrich) in HFE75003M corporation to the barcoded droplets to break the droplets.
Wash the released CAPs three times with TE bufferThermofisher containing 0.1 % (v/v) Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P6585 .
Mix 20 μL dense-packed CAPs containing approximately 9,000 cells with 40 µL 0.8 Molarity (M) NaOH . Incubate at 55 °C 00:10:00 .
10m
Add 40 µL 0.8 Molarity (M) acetic acid to neutralize the solution.
Purify the DNA products using AMPure XP magnetic beads.
Dissolve the purified DNA products in 15 μL nuclease-free water.
Bulk PCR and sequencing
2h
Prepare a 50 µL PCR reaction mix containing:
15 µL purified linear amplification product
25 µL KAPA HiFi HotStart ReadyMixRocheCatalog #07958935001
5 µL forward primer
5 µL reverse primer.
Run the PCR using the following program:
98 °C 00:03:00
16 cycles of:
98 °C 00:00:10
60 °C 00:00:30
72 °C 00:05:00
Final incubation of:
72 °C 00:05:00
4 °C Hold
2h
Purify the PCR product using magnetic beads. Examine the purified PCR product using a Bioanalyzer (Agilent 2100).
Use the purified PCR product to prepare the Nanopore sequencing library with the Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114 and Native Barcoding Kit 24 V14 (SQK-NBD114.24)Oxford Nanopore TechnologiesCatalog #SQK-NBD114.24
Follow the manufacturer’s protocol for Nanopore DNA library preparation.
Sequence the final DNA library on an Oxford Nanopore GridION MK1 with P2 Solo with PromethION Flow Cell (FLO-MIN114).
Equipment
Oxford Nanopore GridION MK1 with P2 Solo
NAME
Flow cell
TYPE
Oxford Nanopore Technologies
BRAND
FLO-MIN114
SKU
Citations
Step 12
Li L, Zhang R, Chen L, Tian X, Li T, Pu B, Ma C, Ji X, Ba F, Xiong C, Shi Y, Mi X, Li J, Keasling JD, Zhang J, Liu Y. Permeability-Engineered Compartmentalization Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems.
https://doi.org/10.1002/advs.202203652Step 12
Li J, Du Y, Shi Q, Chen L, Gao X, Liu Y, Luo Y. Permeability-Engineered Compartmentation System-Enabled Digital PCR (PECS-dPCR): A Digital Platform toward Multistep Biomolecular Assays.
https://doi.org/10.1021/acs.analchem.4c04270Step 44
Du Y, Wen C, Luo Y, Liu Y. Deterministic Co-encapsulation of Microparticles in Droplets via Synchronized Merging for Single-Cell Genomics.
https://doi.org/10.1021/acs.analchem.5c05488