Jul 07, 2025
Pore-C Protocol for Plant Samples
- Melina Sophie Nowak1,
- Boas Pucker2
- 1TU Braunschweig;
- 2Plant Biotechnology and Bioinformatics, IZMB, University of Bonn
- High molecular weight DNA extraction from all kingdomsTech. support email: [email protected]
Protocol Citation: Melina Sophie Nowak, Boas Pucker 2025. Pore-C Protocol for Plant Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9mmrgx1/v1
Manuscript citation:
Nowak et al. 2024, Genome sequence and RNA-seq analysis reveal genetic basis of flower coloration in the giant water lily Victoria cruziana
(https://doi.org/10.1101/2024.06.15.599162)
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: June 28, 2025
Last Modified: July 07, 2025
Protocol Integer ID: 221202
Keywords: Oxford Nanopore, Pore-C, Plants, plant sample, water lily victoria cruziana, pore, end pore, following protocol, protocol, published protocol, workflow
Abstract
The following protocol represents an end-to-end Pore-C workflow tested on the water lily Victoria cruziana. This workflow is an adapted version based on several previously published protocols.
Materials
Note: Material list is intended for one sample.
Day 1: Crosslinking
| Buffer / Chemicals | Materials | Devices | |
| 2.5 M glycine solution | 1x 500 ml Round-bottom flask | Rotovapor or other vacuum device | |
| Vacuum infiltration buffer (400 ml) | Double gloves | Fume hood | |
| 10% ECOSURF EH-9 solution | 1x Büchner funnel | ||
| Formaldehyde | Sintered filter or paper filter | ||
| ddH20 | 1x 500 ml filtering flask | ||
| Spatula | |||
| 1x 50 ml tubes | |||
| Liquid nitrogen (in case of snap freezing) | |||
| Paper towel |
Day 1: Cryogrinding
| Buffer / Chemicals | Materials | Devices | |
| Liquid nitrogen | |||
| Mortar and pestle (Pre-cooled at -80°C) | |||
| Spatula | |||
| 1x chilled 50 ml tube | |||
| Ice | |||
| Styrofoam pad |
Day 1: Nuclei isolation
Note: Homogenisation buffer, nuclei isolation buffer and washing buffer do not require autoclaving.
| Buffer / Chemicals | Materials | Devices | |
| 1x PBS (chilled) | 1x Pre-cooled 50 ml tube (4°C) | Centrifuge (4°C) for 50 ml | |
| 10x Homogenisation buffer (pH 9.0 - 9.4) | Ice | Centrifuge (4°C) for 2 ml | |
| Nuclei isolation buffer (chilled, with β-mercaptoethanol) | Cell strainer (40 µm) | ||
| Washing buffer (chilled) | Wide-bore pipette tips | ||
| 1x pre-cooled 2 ml tubes |
Day 1: Chromatin denaturation
| Buffer / Chemicals | Materials | Devices | |
| 0.5% SDS | Wide-bore pipette tips | Thermomixer (Pre-heated to 62°C, later 37°C) | |
| 10% ECOSURF EH-9 | Cut reaction: NEB CutSmart buffer, NEB Nlall | ||
| Nuclease free water |
Day 2: Proximity ligation (Variant A)
| Buffer / Chemicals | Materials | Devices | |
| Ligation reaction: Nuclease free water, NEB ligase buffer, recombinant albumin, T4 DNA ligase | Thermomixer (Pre-heated to 65°C, later 16°C) | ||
| Wide-bore pipette tips |
Day 2: Protein degradation and de-crosslinking
| Buffer / Chemicals | Materials | Devices | |
| 10% SDS stock solution | Protein degradation reaction: Nuclease free water, Tween-20, SDS (10%), Proteinase K | Thermomixer (Pre-heated to 56°C) |
Day 3: DNA extraction
Note: 5 M NaCl and 3 M sodium acetate solution requires autoclaving. If TE buffer is selfprepared, autoclaving is also required.
| Buffer / Chemicals | Materials | Devices | |
| phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 10 mM Tris-HCL pH 8.0 1 mM EDTA (chilled) | 4x Pre-cooled 50 ml (or 5 ml tubes) | Centrifuge (15°C and 4°C) | |
| 5 M NaCl | Ice | Qubit (dsDNA BR Assay) | |
| 3 M sodium acetate (pH 5.5) | Nuclease free water | ||
| EtOH 100% | 1x 1.5 ml tube | ||
| EtOH 80% | |||
| EtOH 70% | |||
| TE buffer |
Custom buffer for size selection
Note: 0.5 M EDTA pH 8.0, PEG 8000 (40% (w/v)) solution, Tris-HCl 1M and NaCl 5 M requires autoclaving.
| Component | Stock conc. | |
| Tris-HCl | 1 M | |
| EDTA (pH 8.0) | 0.5 M | |
| NaCl | 5 M | |
| PEG 8000 | 40% (w/v) | |
| Nuclease-free water | / |
Size selection
| Buffer / Chemicals | Materials | Devices | |
| Custom buffer with AMPure XP beads | 1.5 ml tube | Magnetic rack | |
| 70% EtOH | Thermomixer (Pre-heated to 50°C) | ||
| TE buffer pH 8.0 | Hula mixer | ||
| Qubit (dsDNA BR Assay) |
Troubleshooting
Day 1: Crosslinking: Preparations
Prepare 10 ml filtered 2.5 M glycine.
Prepare 400 ml fresh vacuum infiltration buffer, store at 4°C prior to use.
| Component | Stock conc. | Final conc. | Volume | |
| PBS pH 7.4 | 10x | 1x | 40 ml | |
| Sucrose | / | 100 mM | 13.69 g | |
| ddH2O | / | / | Up to 400 ml | |
| Total | 400 ml |
Vacuum infiltration buffer for one sample.
Prepare 10% v/v ECOSURFTM EH-9 solution
Day 1: Crosslinking: Procedure
Collect ~2 g plant material. Young, fresh material is recommended. If necessary, material can be disected into smaller pieces.
Wash material with ddH2O and carefully dry with paper towel.
Preload 100 ml of vacuum infiltration buffer in a 500 ml round-bottom flask.
Suspend 2 g of plant material in the preloaded buffer.
Add 100 µl of 10% ECOSURFTM EH-9 to the plant suspension to get a final concentration of 0.01% v/v (Reduces surface tension and prevent plant material from clumping). Perform all subsequent steps inside a fume hood with double gloves.
Add 2910 µl of 36.5% v/v formaldehyde to plant suspension for a final concentration of 1% v/v.
Connect round-bottom flask to a rotovapor and apply 160 mBar (16 kPa) vacuum with 100 rpm for 15 minutes at room temperature.
After 15 minutes, release vacuum to atmospheric pressure and reapply 160 mBar (16 kPa) vacuum with 100 rpm for 15 minutes at room temperature.
Release vacuum to atmospheric pressure and decant suspension into Büchner funnel with filter (sintered filter or paper filter).
Vacuum filter plant suspension over 500 ml filtering flask. Discard filtrate.
Resuspend plant material in 100 ml of fresh vacuum infiltration buffer in the round-bottom flask.
Add 5270 µl of 2.5 M glycine to plant suspension for a final concentration of 1% w/v gylcine and mix by swirling.
Connect round-bottom flask to a rotovapor and apply 160 mBar (16 kPa) vacuum with 100 rpm for 10 minutes at room temperature.
Release vacuum to atmospheric pressure and decant suspension into Büchner funnel with filter (sintered filter or paper filter).
Rinse out round-bottom flask with ddH2O and also decant into Büchner funnel.
Vacuum filter plant suspension over 500 ml filtering flask. Discard filtrate.
Wash plant material twice with 50 ml vacuum infiltration buffer for ~3 minutes in Büchner funnel. Vacuum filter and discard filtrate.
Wash plant material in Büchner funnel with 50 ml distilled water. Filter again. Discard filtrate.
Transfer plant material using a spatula into a fresh 50 ml centrifuge tube.
Now, crosslinked plant material can be removed from the fume hood.
Directly continue to cryogrinding OR snap freeze material in liquid nitrogen and store as -80°C for later use.
Day 1: Cryogrinding: Preparations
Pre-cool mortar and pestle at -80°C for at least 30 minutes.
Get liquid nitrogen.
Cool 50 ml tube.
Day 1: Cryogrinding: Procedure
Place chilled mortar and pestle on styrofoam pad.
Add liquid nitrogen into mortar and then add cross-linked plant material.
Grind material briefly until liquid nitrogen evaporates.
Grind cross-linked plant material into fine powder. Minimize thawing! If material starts thawing, add small volume of liquid nitrogen.
Collect cryo-ground powder into chilled 50 ml tube on ice using a spatula.
Directly continue to nuclei isolation OR snap-freeze powder in liquid nitrogen and then store at -80°C for later use.
NOTE: Do not proceed futher unless it is possible to complete the remaining protocol until size selection.
Day 1: Nuclei isolation: Preparations
Pre-cool 50 ml centrifuge to 4°C
Pre-cool 2 ml centrifuge to 4°C
Cool 1x 50 ml tubes on ice.
Prepare 10 ml of 1x PBS and store at 4°C prior to use.
Prepare fresh 10x homogenization buffer:
| Component | Stock conc. | Final conc. | Volume | |
| Trizma-base | / | 100 mM | 0.61 g | |
| KCl | / | 800 mM | 3.00 g | |
| EDTA | 0.5 M | 100 mM | 10 ml | |
| Spermidine trihydrochloride | / | 10 mM | 0.13 g | |
| Spermine tetrahydrochloride | / | 10 mM | 0.17 g | |
| ddH2O | / | / | Up to 50 ml | |
| Total | 50 ml |
10x homogenization buffer for one sample. NOTE: Adjust pH to 9.0 - 9.4 with NaOH or acetic acid and store at 4°C prior to use.
Prepare fresh nuclei isolation buffer:
| Component | Stock conc. | Final conc. | Volume | |
| 10x homogenization buffer | 10x | 1x | 10 ml | |
| Sucrose | / | 500 mM | 17.12 g | |
| PVP-40 | / | 1% w/v | 1.00 g | |
| ECOSURF EH-9 | 100% | 0.5% v/v | 500 µl | |
| ddH2O | / | / | Up to 100 ml | |
| β-mercaptoethanol | 100% (14.3 M) | 0.25% v/v (35.75 mM) | 250 µl | |
| Total | 100 ml |
Nuclei isolation buffer for one sample. NOTE: Do not add β-mercaptoethanol until ready to use. Store at 4°C prior to use.
Prepare fresh washing buffer:
| Component | Stock conc. | Final conc. | Volume | |
| 10x homogenization buffer | 10x | 1x | 1 ml | |
| Sucrose | / | 500 mM | 1.71 g | |
| ddH2O | / | / | Up to 10 ml | |
| Total | 10 ml |
Washing buffer for one sample. NOTE: Store at 4°C prior to use.
Day 1: Nuclei isolation: Procedure
Resuspend cryo-ground plant material in 20 ml of chilled nuclei isolation buffer in a 50 ml tube. Note: Buffer has to be supplemented with β-mercaptoethanol!
Carefully inverting the tube by hand for 15 minutes.
Pass the plant suspension through a 40 µm cell strainer into a fresh chilled 50 ml tube on ice. This step might take a while. If cell strainer is clogged, transfer samples to a new cell strainer and continue.
After the sample has passed the strainer, rinse the original sample tube with further 5 ml of chilled nuclei isolation buffer and filter through the (same) 40 µm cell strainer.
Centrifuge for 20 minutes at 4°C with a centrifugation force recommended for the genome size:
| Genome size (Mbp) | Centrifugation force (g) | |
| 25 | 4800 | |
| 50 | 4370 | |
| 100 | 3970 | |
| 250 | 3500 | |
| 500 | 3190 | |
| 1 000 | 2900 | |
| 2 500 | 2560 | |
| 5 000 | 2330 | |
| 10 000 | 2120 | |
| 25 000 | 1870 | |
| 50 000 | 1700 |
Centrifugation forces according to genome size. Based on: "Restriction enzyme Pore-C protocol for plant samples" v5, 02nd December 2024.
Carefully discard supernatant.
Using a wide-bore pipette tip, gently resuspend the pellet in 1 ml chilled nuclei isolation buffer.
Add further 19 ml chilled nuclei isolation buffer and mix by swirling.
Centrifuge for further 10 minutes at 4°C using a centrifugation force appropriate to genome size (see table).
Repeat step 44 - 47 from this section.
Pre-cool 2 ml centrifuge to 4°C.
Carefully discard supernatant.
Using a wide-bore pipette tip, carefully resuspend the pellet in 1 ml cold washing buffer.
Transfer sample to fresh 2 ml on ice.
Rinse original tube with further 1 ml of cold washing buffer and transfer to the same tube as the transferred sample.
Centrifuge for 5 minutes at 4°C using a centrifugation force recommended for the genome size (see table).
Discard as much supernatant as possible without disturbing the pellet.
Gently resuspend the pellet in 2 ml of chilled 1x PBS using a wide-bore pipette tip.
Centrifuge for 5 minutes at 4°C at 3000 g.
Discard supernatant.
Day 1: Chromatin denaturation: Preparations
Pre-heat thermomixer to 62°C, later to 37°C.
Prepare 0.5% SDS dilution.
Day 1: Chromatin denaturation: Procedure
Resuspend pellet of crosslinked nuclei in 100 µl of 0.5% SDS and mix by gentle pipetting with a wide-bore pipette tip.
Incubate at 62°C for 6 minutes without agitation. Then cool to room temperature.
Pre-heat thermomixer to 37°C.
Directly add 250 µl nuclease-free water and 50 µl of 10% v/v ECOSURFTM EH-9 to the sample.
Mix gently by pipetting with wide-bore pipette tip.
Incubate at 37°C for 15 minutes without agitation. Chromatin is now ready for digestion.
Add following reagents to the sample for final concentration of 1 U/µl for the selected restriction enzyme (Nlalll):
| Component | Stock conc. | Final conc. | Volume | |
| Plant nuclei suspension | / | / | 400 µl | |
| NEB CutSmart buffer | 10x | 1x | 50 µl | |
| NEB Nlall | 10 U/µl | 1 U/µl | 50 µl | |
| Total | 500 µl |
Restriction enzyme mix for one sample.
Mix by gentle inversion and incubate sample in the thermomixer at 37°C for 18 hours without agitation.
Day 2: Proximity ligation: Preparations
NOTE: Proximity ligation is depending on the previous chosen restriction enzyme for digestion (enzyme heat-denaturable or not). The following steps are applicable for NIaIII. This enzyme is recommended by ONT as it is suitable for many species and generates high density contact outputs with optimal fragment length.
However, an in silico restriction digest can be performed to identify potential areas of reduced cleavage within the genome or repeat rich genomic regions to determine other restriction enzyme candidates.
Pre-heat thermomixer to 65°C, later 16°C.
Day 2: Proximity Ligation: Procedure
Heat-denature the restriction enzyme by incubating the sample in the thermomixer at 65°C for 20 minutes at 300 rpm.
Pre-cool thermomixer to 16°C.
Allow sample to cool to room temperature.
Set up proximity ligation by adding the reagents in the following order directly to the sample:
| Component | Stock conc. | Final conc. | Volume | |
| Plant nuclei suspension (Digestion reaction) | / | / | 500 µl | |
| Nuclease-free water | / | / | 345 µl | |
| NEB ligase buffer | 10x | 1x | 100 µl | |
| Recombinant albumin | 20 µg/µl | 0.1 µg/µl | 5 µl | |
| T4 DNA ligase | 400 U/µl | 20 U/µl | 50 µl | |
| Total | 1000 µl |
Proximity ligation set up for one sample.
Mix by gentle pipetting with wide-bore pipette tip and incubate the sample in a thermomixer at 16°C for 6 hours without agitation. Note: Avoid prolonged ligation to minimize trans-chromosomal contacts.
Day 2: Protein degradation and de-crosslinking: Preparations
Pre-heat thermomixer to 56°C.
Prepare 10% SDS stock solution.
Day 2: Protein degradation and de-crosslinking: Procedure
Set up protein degradation reaction by adding the following reagents to the ligation reaction:
| Component | Stock conc. | Final conc. | Volume | |
| Plant nuclei suspension (Ligation reaction) | / | / | 1000 µl | |
| Nuclease-free water | / | / | 300 µl | |
| Tween-20 | 20% | 5% | 500 µl | |
| SDS | 10% | 0.5% | 100 µl | |
| Proteinase K | 20 µg/µl | 1 µg/µl | 100 µl | |
| Total | 2000 µl |
Protein degradation mix for one sample.
Mix through gentle inversion.
Incubate sample in a thermomixer at 56°C for 18 hours with periodic <1000 rpm for <30 seconds every 15 minutes (to prevent condensation).
Day 3: DNA extraction: Preparations
Pre-cool centrifuge to 15°C.
Cool 50 ml tubes (if available 5 ml tubes).
Prepare 5 M NaCl solution.
Prepare 3 M sodium acetate (pH 5.5).
Prepare EtOH dilutions: 100%, 80%, 70%
Prepare TE buffer.
Day 3: DNA extraction: Procedure
Cool sample on ice.
Transfer entire volume to a 50 ml tube (or 5 ml tube).
Rinse original tube with 200 µl of nuclease-free water and transfer to the 50 ml tube (or 5 ml tube). Total volume should be ~2200 µl.
Add the same volume of chilled phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 10 mM Tris-HCL pH 8.0 1 mM EDTA. Adjust volume accordingly to equal the sample volume.
Mix through gentle inversions for 5 minutes. A homogenous emulsion should be obtained.
Centrifuge sample at 15 000 g at 15°C for 15 minutes.
Carefully collect ~2000 µl of the upper aqueous phase and transfer to a fresh chilled 50 ml tube (or 5 ml tubes).
Add the same volume of chilled phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 10 mM Tris-HCL pH 8.0 1 mM EDTA. Adjust volume accordingly to equal the sample volume.
Mix through gentle inversion for 5 minutes. A homogenous emulsion should be obtained.
Centrifuge sample at 15 000 g, 15°C for 15 minutes.
Carefully transfer the aqueous phase to a fresh chilled 50 ml tube (or 5 ml tube) and note the recovered volume (~1000 µl). Note: Do not take any of the interphase layer.
Split the recovered aqueous phase to a second 50 ml tube (or 5 ml tubes) to get two equal aliquots in two seperate tubes.
Add 0.02 volumes of 5 M NaCl (0.1 M final) and 0.1 volumes of 3 M sodium acetate pH 5.5 (0.3 M final) based on the volume of the recovered aqueous phase in step 97 to both 50 ml tubes (or 5 ml tubes).
Mix through gentle agitation.
Add 3 volumes of 100% EtOH based on the volume of the recovered aqueous phase in step 97 to both 50 ml tubes (or 5 ml tubes).
Mix thorugh gentle inversion and precipitate at -80°C for 3h - 6h. This time span resulted in positive results for Victoria cruziana. However, ONT recomments at least >1h or overnight at -20°C.
Pre-cool centrifuge to 4°C.
Centrifuge the samples in the 50 ml tubes (or 5 ml tubes) at 16 000 g at 4°C for 30 minutes.
Discard the supernatant before washing the pellets in 4 ml 80% EtOH.
Centrifuge both samples at 16 000 g at 4°C for 5 minutes.
Discard supernatant before washing the pellets in 2 ml 70% EtOH.
Centrifuge both samples at 16 000 g at 4°C for 5 minutes.
Discard the supernatant.
Briefly spin down the tubes and remove any residual supernatant.
Air-dry the pellets for 5 minutes.
Gently resuspend each samples in 75 µl TE or LTE buffer and incubate for 5 minutes at toom temperature. Mix through gentle agitation every few minutes.
Briefly spin down the tubes and pool both samples into a fresh 1.5 ml tube.
Quantify the DNA concentration by using a Qubit Assay Kit and measure dsDNA. Samples might be diluted 1/10 as Qubit reading could be affected by high salt concentrations.
Store the samples at 4°C until library preparation.
Size selection for Pore-C samples
NOTE: The following part is based on a protocol from Miriam Schalamun and Benjamin Schwessinger, with some modifications.
To enrich >2 kb fragments for optimal Pore-C data, it is recommended to use 0.85x volume of custom SPRI beads (AMPure XP Beads).
Size selection: Preparation of custom buffer with AMPure XP beads
Prepare PEG 8000 Stock solution 40% w/v: 40g / 100 ml for 1 ml buffer. Take 0.4 g of PEG 8000.
Pipette custom buffer for size selection:
NOTE: Accuratly pipette 548 µl of 40% (w/v) PEG 8000 using a wide-bore pipette tip.
| Component | Stock conc. | Final conc. | Volume | |
| Tris-HCl | 1 M | 10 mM | 20 µl | |
| EDTA pH 8.0 | 0.5 M | 1 mM | 4 µl | |
| NaCl | 5 M | 1.6 M | 640 µl | |
| PEG 8000 | 40% (w/v) | 11% (w/v) | 548 µl | |
| Nuclease-free water | / | / | 780 µl | |
| Total | 1992 µl |
Preparation of custom buffer for size selection.
Transfer mixed AMPure XP beads into two 1.5 ml tubes (each containing 1 ml).
Place tubes on magnetic rack until solution is clear. The tube lid should be opened before placing the tube on the magnetic rack to avoid disturbing the sedimented beads at a later stage.
Discard supernatant.
Remove tubes from magnetic rack. Wash beads by resuspending the pellet in 1 ml of nuclease-free water.
Place tubes on the magentic rack again with open lid and allow beads to pellet.
Pipette off the supernatant.
Repeat step 122 and 124.
Spin down and place the tubes back on the magnetic rack. Remove any residual water using a pipette.
Resuspend the bead pellets in both tubes with 200 µl custom buffer.
Pool both resuspended pellets into the remaining custom buffer.
If not used immediatly, store at 4°C. After storing the custom buffer at 4°C bring the bead suspension back to room temperature before use.
Size selection: Procedure
Pre-heat thermomixer to 50°C.
Dilute sample DNA to 60 ng/µl in a final volume of 50 µl TE buffer pH 8.0.
Add 0.85x (42.5 µl) of the custom bead suspension to the DNA sample.
Mix by flicking the tube and incubate for 10 minutes on a Hula mixer at room temperature.
Spin down briefly and pellet on a magnetic rack.
Keep tube on the magnet and pipette off the supernatant.
Keep tube on the magnetic rack and wash beads with 200 µl 70% EtOH (prepared fresh) without disturbing the pellet.
Remove the 70% EtOH and discard.
Repeat step 136 and 137 again.
Spin down the tube and place it back on the magnetic rack. Pipette off any residual volume.
Air-dry the pellet for 30 sec.
Remove the tube from the magnetic rack and resuspend the pellet in 40 µl TE buffer.
Incubate for 1 minute at 50°C and afterwards for 5 minutes at room temperature.
Pellet the beads on the magnetic rack until the eluate is clear.
Pipette off 40 µl of eluate into a clean 1.5 ml tube.
Quantify 1 µl of size-selected DNA using a Qubit fluorometer. Expect around 50% loss of DNA through size selection.
Information: Library preparation
For library preparation of pore-c DNA, the protocol of the ligation sequencing kit V14 (SQK-LSK114) has been used with the following deviations:
- Step 6: Incubation in thermal cycler at 20°C for 15 minutes then 65°C for 5 minutes
(instead of 20°C for 5 minutes and 65°C for 5 minutes).
Information: Bioinformatic analysis
For further informations regarding bioinformatic analysis see: https://doi.org/10.1101/2024.06.15.599162
Protocol references
This workflow is an adapted version based on several previously published protocols:
“Restriction enzyme Pore-C protocol for plant samples” v5, 02nd December 2024
(https://nanoporetech.com/document/extraction-method/plant-pore-c)
Belaghzal, H., Dekker, J. and Gibcus, J. H. (2017) Hi-C 2.0: an optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation, Methods, 123, pp. 56–65. doi: 10.1016/j.ymeth.2017 .04.004.HI-C.
Belton, J.-M. et al. (2012) Hi-C: a comprehensive technique to capture the conformation of genomes, Methods, 58(3), pp. 1–16. doi: 10.1016/j.ymeth.2012.05.001.Hi-C.
Folta, K. M. and Kaufman, L. S. (2006) ‘Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays’, Nature Protocols, 1(6), pp. 3094–3100.
Gavrilov, A. A., Golov, A. K. and Razin, S. V . (2013) Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure, PLoS ONE, 8(3), pp. 1–6. doi: 10.1371/journal.pone.0060403.
Lieberman-Aiden, E. et al. (2009) Comprehensive mapping of long range interactions reveals folding principles of the human genome, Science, 326, pp. 289–293.
Liu, C. (2017) ‘Plant Gene Regulatory Networks’, in Plant Gene Regulatory Networks: Methods and Protocols, Methods in Molecular Biology, pp. 155–166. doi: 10.1007/978-1-4939-7125-1.
Nagano, T . et al. (2015) Comparison of Hi-C results using in-solution versus in-nucleus ligation, Genome Biology, 16(1), pp. 1–13. doi: 10.1186/s13059-015-0753-7 .
Ulahannan, N. et al. (2019) Nanopore sequencing of DNA concatemers reveals higher-order features of chromatin structure, bioRxiv, p. 833590; doi: 10.1101/833590.
Workman, R. et al. (2018) ‘High molecular weight DNA extraction from recalcitrant plant species for third generation sequencing’, Protocol Exchange, version 1, pp. 1–15. doi: 10.1038/protex.2018.059.
Zhang, M. et al. (2012) ‘Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research’, Nature Protocols. Nature Publishing Group, 7(3), pp. 467 –478. doi: 10.1038/nprot.2011.455.
“SPRI size selection protocol for >1.5-2 kb DNA fragments”; based on the work by Miriam Schalamun and Benjamin Schwessinger, with some modifications
(https://nanoporetech.com/document/extraction-method/spri-size-selection)
For further detailed information on alternative applications, please consider the original protocol: “Restriction enzyme Pore-C protocol for plant samples” (ONT).
Nowak et al. 2024, Genome sequence and RNA-seq analysis reveal genetic basis of flower coloration in the giant water lily Victoria cruziana
(https://doi.org/10.1101/2024.06.15.599162)
Acknowledgements
We thank all members of the research group Plant Biotechnology and Bioinformatics.