Oct 05, 2022
BIND&MODIFY: Long-range single-molecule mapping of chromatin modification in eukaryotes. V.2
- Zhe Weng1,
- Fengying Ruan1,
- Weitian Chen1,
- Zhichao Chen1,2,
- Yeming Xie1,
- Meng Luo1,
- Chen Zhang1,
- Zhe Xie1,
- Chen Tan1,
- Juan Wang1,
- Yuxin Sun1,
- Yitong Fang1,
- Mei Guo1,
- Hongqi Wang1,
- Chong Tang1,3,4
- 1BGI Genomics, BGI-Shenzhen, Shenzhen 518083, China;
- 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China;
- 3Nantong University, Nantong, 226000, China;
- 4Nephrosis Precision Medicine Innovation Center, University of Beihua School of Medicine, Jilin, 132011, China
External link: https://doi.org/10.1186/s13059-023-02896-y
Protocol Citation: Zhe Weng, Fengying Ruan, Weitian Chen, Zhichao Chen, Yeming Xie, Meng Luo, Chen Zhang, Zhe Xie, Chen Tan, Juan Wang, Yuxin Sun, Yitong Fang, Mei Guo, Hongqi Wang, Chong Tang 2022. BIND&MODIFY: Long-range single-molecule mapping of chromatin modification in eukaryotes. V.2. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4j7m8lo5/v1
Manuscript citation:
Weng Z, Ruan F, Chen W, Chen Z, Xie Y, Luo M, Xie Z, Zhang C, Wang J, Sun Y, Fang Y, Guo M, Tan C, Chen W, Tong Y, Li Y, Wang H, Tang C, BIND&MODIFY: a long-range method for single-molecule mapping of chromatin modifications in eukaryotes. Genome Biology doi: 10.1186/s13059-023-02896-y
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: In development
We are still developing and optimizing this protocol
Created: October 05, 2022
Last Modified: October 05, 2022
Protocol Integer ID: 70841
Keywords: Epigenetics, Transcription Factor, Histone Modification, Single Molecule Sequencing, ONT
Funders Acknowledgement:
Science, Technology, and Innovation Commission of Shenzhen Municipality
Grant ID: JSGG20170824152728492
Disclaimer
The authors declare no competing interests.
Abstract
Here we describe a powerful method, BIND&MODIFY, for probing histone modifications and transcription factors at single molecular level. This is the Protocol V2. Our approach used the recombinant fused protein A-M.EcoGII, which tethers the methyltransferase M.EcoGII to the protein binding sites and locally labels the neighboring DNA regions via artificial methylations. This method could reveal ingle-molecule heterogenous histone modification status and CpG methylation at the same time, and could enable quantify the correlation between the distal elements. Further applications based on this method's concept could be applied to probe multiple protein binding events on the same single molecular DNA. The method proposed herein may soon become an essential tool for third-generation sequencing in the future.
Materials
1.5ml DNA LoBind tubes (Eppendorf, 0030108051)
Digitonin (Abcam, ab141501)
Roche Complete Protease Inhibitor EDTA-Free tablets (Sigma, 5056489001)
Bovine Serum Albumin (BSA), 20mg/ml, (NEB, B9000S)
Spermidine (Sigma, S0266)
Glysine (Sigma, 50046)
Pierce 16% Formaldehyde (w/v)（Thermo Scientific, 28908)
Polyvinylpyrrolidone, MW 40,000 (Sigma, PVP40)
Sodium metabisulfite (Sigma, 9000)
Polyethelene Glycol 8000(PEG) (Fisher Scientific, BP233-100)
RNase A, 10mg/ml (Thermo Scientific, EN0531)
Phenol:Chloroform:Isoamyl Alcohol 25:24:1 (Sigma, P3803)
S-adenosylmethionine (SAM), 32mM (NEB, B9003S)
Concanavalin A (ConA)-coated magnetic beads (Bangs Laboratories, BP531)
Sera-mag SpeedBeads carboxylate modified magnetic particles, 5% solids, 50 mg/mL, Hydrophobic, (VWR, 10204-666) (GE Healthcare Life Sciences, 44152105050350)
DNase/RNase-Free Distilled Water (Invitrogen 10977)
KCl, 2M, Rnase free (Invitrogen, AM9640G)
NaCl, 5M, Rnase free (Invitrogen, AM9759)
CaCl2, 1M (Sigma, 21115)
MnCl2, 1M (Sigma, M1787)
Tris, 1M, pH 8.0 (Thermoscientific, AM9856)
HEPES, 1M, pH 7.5 (Thermoscientific, J60712AK)
Potassium acetate, 3M, pH 5.5 (Invitrogen, AM9610)
Sodium acetate, 3M, pH5.2 (Invitrogen, R1181)
Triton X-100 (Sigma, X100)
EDTA, 0.5M, pH 8.0 (Invitrogen, 15575)
SDS, 10% (Invitrogen, 15553)
Tween-20 (Sigma, P9416)
Primary antibody, targeting histone modification and DNA binding protein. For example, α-H3K27me3 rabbit monoclonal antibody (CST, 9733), α-CTCF rabbit polyclonal antibody (Millipore, 07-729-25ul).
Secondary antibody, Guinea Pig anti-Rabbit IgG (Heavy & Light Chain) antibody (Antibodies-Online, ABIN101961).
pA-M.EcoGII Plasmid Amino Acid Sequence:
SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKDDDKEFMLNTVKISSCELINADCLEFIRSLPENSVDLIVTDPPYFKVKPEGWDNQWKGDDDYLKWLDQCLAQFWRVLKPAGSLYLFCGHRLASDIEIMMRERFSVLNHIIWAKPSGRWNGCNKESLRAYFPATERILFAEHYQGPYRPKDAGYEAKGRALKQHVMAPLIAYFRDARAALGITAKQIADATGKKNMVPHWFSASQWQLPNESDYLKLQSLFARVAEEKHQRGELEKPHHQLVSTYSELNRKYMELLSEYKNLRRYFGVTVQVPYTDVWTYKPVQYYPGKHPCEKPAEMLQQIISASS
pA-M.EcoGII Plasmid DNA Expression Sequence:
AGCTTAAAAGATGACCCAAGCCAAAGTGCTAACCTATTGTCAGAAGCTAAAAAGTTAAATGAATCTCAAGCACCGAAAGCGGATAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAACGAAGAACAACGCAATGGTTTCATCCAAAGCCTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCTCAAGCACCAAAAGCTGACAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATTTTACATTTACCTAACTTAACTGAAGAACAACGTAACGGCTTCATCCAAAGCCTTAAAGACGATCCTTCAGTGAGCAAAGAAATTTTAGCAGAAGCTAAAAAGCTAAACGATGCTCAAGCACCAAAAGATGACGATAAAGAATTCATGCTTAATACTGTAAAAATATCCAGTTGTGAGTTAATCAACGCCGACTGCCTGGAATTTATCCGGTCGTTACCCGAAAATTCTGTTGACCTGATAGTCACGGACCCGCCGTACTTTAAAGTGAAGCCCGAGGGCTGGGATAACCAGTGGAAGGGCGACGATGATTACCTGAAGTGGCTGGACCAGTGTCTGGCGCAGTTCTGGCGGGTGCTGAAACCTGCCGGAAGTCTTTACCTGTTCTGTGGCCATCGCCTGGCATCTGACATTGAAATCATGATGCGTGAACGCTTCAGTGTGCTGAACCATATTATCTGGGCAAAGCCGTCCGGACGCTGGAACGGGTGCAACAAGGAAAGCCTGCGGGCGTATTTCCCCGCCACAGAGCGCATTCTGTTCGCGGAACATTATCAGGGGCCGTATCGCCCGAAAGATGCCGGGTATGAGGCGAAGGGCAGGGCACTGAAACAGCATGTGATGGCCCCGCTGATTGCTTACTTTCGTGATGCGCGCGCTGCCCTGGGGATAACGGCAAAACAGATTGCAGATGCCACAGGAAAGAAAAACATGGTGCCGCACTGGTTCAGTGCCAGTCAGTGGCAGCTACCGAACGAAAGCGATTATCTGAAATTACAGTCGCTGTTTGCCCGGGTGGCAGAAGAGAAACATCAGCGCGGGGAACTGGAAAAGCCACACCACCAGCTGGTCAGCACATACAGTGAGCTGAACCGGAAGTATATGGAACTGCTGAGTGAATATAAAAATTTGCGGCGGTATTTCGGTGTGACGGTGCAGGTGCCGTACACCGATGTGTGGACGTATAAACCGGTGCAGTACTATCCAGGGAAACATCCGTGCGAAAAACCGGCAGAAATGCTGCAGCAGATAATCAGCGCAAGTAGTCGTCCTGGTGATCTGGTTGCGGATTTTTTCATGGGGTCGGGTTCAACGGTAAAAGCGGCGATGGCACTGGGGCGTCGTGCGATTGGTGTTGAGCTGGAGACCGGACGTTTTGAGCAGACAGTCAGGGAAGTTCAGGATTTAATCGTT
OVERVIEW
OVERVIEW
This protocol describes step-by-step guidelines for BIND&MODIFY method.
Protocol v2.
BIND&MODIFY method is based on the indirect labelling of DNA regions bound to the protein of interest (with antibody) using an engineered recombinant fusion protein, protein A-M.EcoGII (pA-M.EcoGII), whose methyltransferase activity can be locally controlled.Firstly, BIND&MODIFY method shows comparable distribution of histone modifications (H3K27me3, H3K4me3) and DNA binding protein (CTCF) with conventional ChIP-seq method. Secondly, BIND&MODIFY method resolves histone modification in complex genomic region, phases the epigenome, and uncovers epigenomic heterogeneity at single molecular level. Furthermore, BIND&MODIFY method reveals long-distance correlation between genome regulators. We believe BIND&MODIFY method to become one powerful tool for probing DNA binding protein and their regulatory mechanisms in the upcoming long-read sequencing technology arsenal.
With regard to previous published version,
BIND&MODIFY: Long-range single-molecule mapping of chromatin modification in eukaryotes
To increase the methylation effiency, the following changes were applied and used for targeting CTCF for experiments.
The major changes were:
(1) introduce Protein A block step before antibody binding;
(2) reduce primary antibody binding time from overnight to 2h at 4 degree;
(3) add 0.5mM spermidine in the methylation buffer;
(4) increase of methylation reaction time from 30min to 120min, and replenish 32mM SAM at 30min, 60min, 90min;
(5) add 1ng/ul dsDNA 1ng/ul in the activation buffer(optional).
We refer this BIND&MODIFY protocol as Protocol v2
Figure1 Overview of BIND&MODIFY method
REAGENT SETUP
REAGENT SETUP
2h
2h
Digitonin (5%): Dissolve 100mg digitonin in 2 ml DMSO. Aliquote at 50ul per PCR tube and freeze at -20. Avoid freeze-thaw cycles.
Caution: Digitonin is toxic and avoid any direct contact with skin or during breath. Use full PPE including a mask, lab coat and gloves while handling with digitonin. DMSO can penetrate through the skin.
Binding Buffer: Prepare fresh and store the buffer at 4°C for 6 months.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | 1M HEPES pH 7.9 | 200ul | 20mM | |
| 3 | 1M KCl | 100ul | 10mM | |
| 4 | 1M CaCl2 | 10ul | 1mM | |
| 5 | 1M MnCl2 | 10ul | 1mM | |
| 6 | H2O | 9750ul | ||
| 7 | Total | 10ml |
Wash Buffer: Prepare fresh, and store the buffer at 4°C up to 1 week.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | 1M HEPES pH 7.5 | 1.0ml | 20mM | |
| 3 | 5M NaCl | 1.5ml | 150mM | |
| 4 | 2M Spermidine | 12.5ul | 0.5mM | |
| 5 | Roche Proteinase Inhibitor cocktail tablet | 1 tablet | 1X | |
| 6 | H2O | 47.5ml | ||
| 7 | Total | 50ml |
Dig-wash Buffer: Prepare fresh, store the buffer at 4°C up to 2 days.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | 2% Digitonin | 400ul | 0.02% | |
| 3 | 20% BSA | 200ul | 0.1% | |
| 4 | 1X Wash Buffer | 39.6ml | ||
| 5 | Total | 40ml |
Tween-wash Buffer: Prepare fresh, store the buffer at 4°C up to 2 days.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | 1M HEPES-KOH, pH 7.5 | 1000ul | 20mM | |
| 3 | 5M NaCl | 1500ul | 150mM | |
| 4 | 2M Spermidine | 12.5ul | 0.5mM | |
| 5 | Roche Proteinase Inhibitor cocktail tablet | 1 tablet | 1X | |
| 6 | 20% BSA | 250ul | 0.1% | |
| 7 | 100% Tween-20 | 50ul | 0.1% | |
| 8 | Total | 50ml |
Methylation Buffer: Prepare fresh. Add SAM only before the methylation reaction.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | 10X CutSmart Buffer | 30ul | 1x | |
| 3 | 50X Roche Proteinase Inhibitor cocktail | 6ul | 1x | |
| 4 | 5% Digitonin | 3ul | 0.05% | |
| 5 | 32mM SAM | 7.5ul | 800uM | |
| 6 | 20% BSA | 1.5ul | 0.10% | |
| 7 | 2M Spermidine | 0.125ul | 0.05mM | |
| 8 | 10ng/ul dsDNA(optional) | 1ul | 1ng/ul | |
| 9 | H2O | 250ul | ||
| 10 | Total | 300ul |
Digestion Buffer: Prepare fresh.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | Polyvinylpyrrolidone 40 | 0.1g | 1% | |
| 3 | Sodium metabisulfite | 0.1g | 1% | |
| 4 | 5M NaCl | 1.0ml | 0.5M | |
| 5 | 1M Tris-HCl, pH 8.0 | 1.0ml | 0.5M | |
| 6 | 0.5M EDTA | 1.0ml | 50mM | |
| 7 | 20% SDS | 625ul | 1.25% | |
| 8 | H2O | 6375ul | ||
| 9 | Total | 10ml |
Mix and incubate at 65°C during at least 30 minutes. The solution need to be clear before use.
Serapure Beads Solution: Store the solution at 4°C for 1 month.4ml serapure beads wash 4 times with water to remove sodium azide,then resuspend in 10ml serapure beads solution.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | 50% PEG8000 | 3.6 ml | 18% | |
| 3 | 5M NaCl | 2 ml | 1M | |
| 4 | 1M Tris-HCl, pH 8.0 | 0.1ml | 10mM | |
| 5 | 0.5M EDTA | 20ul | 1mM | |
| 6 | 100% tween 20 | 5ul | 0.05% | |
| 7 | H2O | 4275ul | ||
| 8 | Total | 10ml |
Bead Binding Buffer: Store the solution at 4°C for 1 week.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | 50% PEG 8000 | 4ml | 20% | |
| 3 | 5M NaCl | 6ml | 3.0M | |
| 4 | Total | 10ml |
Mix until the solution becomes clear. If PEG8000 is not dissolved, it can lead to a poor yield because PEG8000 makes gDNA to bind to the beads.
Bead Washing Buffer: Prepare fresh.
| A | B | C | D | |
| A | B | C | ||
| 1 | Reagent | Quantity | Final Concentration | |
| 2 | Absolute Ethanol | 35ml | 70% | |
| 3 | H2O | 15ml | ||
| 4 | Total | 50ml |
Cells Preparation
Cells Preparation
1h 30m
1h 30m
Harvest fresh cells at room temperature.
Note: Use Eppendorf DNA LowBind tube during the whole protocol to reduce cell/DNA loss.
Centrifuge cells at 300g for 5 min at 4°C.
Resuspend the cells in 1ml cold PBS, repeat step 5-step 6 twice.
Resuspend the cells in 900ul cold PBS. Count the cells. For lightly fixed cells, go to step 7.
Normally this protocol works for 5x10^5 cells per methylation reaction. Aliquot 4x10^6 cells per centrifuge tube for 8 tube methylation reaction.
Add freshly prepared 1% formaldehyde into the resuspended cells (100ul into 900ul cells), and put at room temperature for 10 min.
Stop the crosslinking by adding 1.25M glycine to twice molar ratio of formaldehyde (60ul into 1ml cell fixing reaction).
Centrifuge cells at 500g for 5 min at 4°C. Carefully remove all the liquids from the supernatant with 1000ul pipette tip followed by 100ul and 10ul pipette tip.
Resuspend the cells with Wash Buffer and count the fixed cells.
Normally this protocol works for 5x10^5 cells per methylation reaction. Aliquot 4x10^6 cells per centrifuge tube for 8 tube methylation reaction.
Bind cells or nuclei to Concanavalin A-coated beads
Bind cells or nuclei to Concanavalin A-coated beads
30m
30m
Gently vortex and resuspend the ConA beads slurry, 10ul of the ConA beads would be enough for 5x10^5 cells. The following is for 4 samples.
Aliquot 90ul ConA beads slurry into 1ml Binding Buffer in a 1.5ml tube and mix by pipetting. Put the tube on a magnetic stand to clear (1-2min).
Remove the liquid completely on the magnetic stand. Add 1ml Binding Buffer and mix by pipetting. Quick spin the tube to remove the liquid from the cap.
Put the tube on a magnetic stand to clear, remove the liquid, and resuspend in 90ul Binding Buffer (10ul per sample) and place the activated beads slurry at room temperature until cells are prepared.
Carefully add in 90ul Binding Buffer containing ConA beads into the tube containing 4x10^6 cells prepared from step 6 or step 10. Place on end-over-end rotator for 10min.
Centrifuge cells at 500g for 5 min at 4°C. Carefully remove all the liquids from the supernatant with 1000ul pipette tip followed by 100ul and 10ul pipette tip.
Carefully add in 1ml Dig-wash Buffer to each of the tubes, place the tubes on ice for 5min. Check 10ul of the cells with Trypan Blue staining. If the cell permeabilization was successful, continue to the next steps.
Protein A Block
Protein A Block
2h
2h
Quick spin the tube to remove the liquid from the cap. Place the tube on a magnetic stand to clear, remove the liquid.
Resuspend the cells in 200ul ice cold Tween-wash Buffer with gentle vortexing.
Add 2.5ul 50ug/ul protein A. Place on end-over-end rotator at room temperature for 10min.
Primary Antibody Binding
Primary Antibody Binding
2h
2h
Quick spin the tube to remove the liquid from the cap. Place the tube on a magnetic stand to clear, remove the liquid.
Resuspend the cells in 400ul ice cold Tween-wash Buffer with gentle vortexing. Divide into eight 1.5ml Eppendorf LowBind tubes, and 50ul each tube. Scale up or down based on specific applications.
Add 0.5-1.0ul of specific primary antibody (H3K27me3 or CTCF) into each tube with gentle vortexing.
Note: We use 1:50-1:100 primary antibody dilution as recommended by CUT&TAG protocol.
Place the tube on end-over-end rotator at 4°C for 2h.
Quick spin the cellls with primary antibody. Place the cells with primary antibody on a magnetic stand, carefully remove the solution.
Add 1ml Tween-wash Buffer. Invert the tube 10 times to resuspend the beads.
Repeat step 23-25 twice.
pA-M.EcoGII Binding
pA-M.EcoGII Binding
1h
1h
Add 10ul pA-M.EcoGII recombinant enzyme into 190ul Tween-wash Buffer, mix gently by pipetting.
Quick spin the tube from step 28. Place the tube on a magnetic stand, carefully remove the solution.
Add the pA-M.EcoGII containing buffer to the cells with gentle vortexing.
Place the tube on end-over-end rotator at room temperature for 1h.
Quick spin the cells with pA-M.EcoGII. Place the cells with pA-M.EcoGII on a magnetic stand, carefully remove the solution.
Add 1ml Tween-wash Buffer. Invert the tube 10 times to resuspend the beads.
Repeat step 30-31 twice.
Methyltransferase Activation
Methyltransferase Activation
2h
2h
Quick spin the tube from step 34. Place the tube on a magnetic stand, carefully remove the solution.
Add 300ul Methylation Buffer. Invert the tube 10 times to resuspend the beads.
Incubate at 37°C thermomixer at 1000rpm for 120min. Supplement 7.5ul 32mM SAM at 30min, 60min, and 90min. Proceed to step 38 with DNA extraction by PCI, or proceed to step 46 with DNA extraction by Serapure Beads.
DNA extraction 1:PCI
DNA extraction 1:PCI
2h
2h
Take the tube from step 37. To stop the methylation reaction, add 10ul 0.5M EDTA, 1.5ul 20% SDS, and 5.0ul 20mg/ml Proteinase K to each tube.
Mix by vortexing at highest speed for 5s. Incubate at 55°C water batch overnight until the solution is clear.
Note: Increase incubation time if the solution is viscous or cloudy.
Add 300ul PCI and vortexing at highest speed for 30s. Invert the tube 10 times to mix thoroughly. Centrifuge at 16000g for 5min.
Transfer the upper liquid aqueous phase to a new 1.5ml centrifuge tube. Add 300ul chloroform. Invert the tube 10 times. Centrifuge at 16000g for 5min.
Transfer the upper liquid aqueous phase to a new 1.5ml centrifuge tube. Add 1/10 volume of 3M sodium acetate solution.
Add 2.5x volume of ice cold 100% ethanol in the solution to precipitate DNA. Incubate the tube at -20 overnight.
Centrifurge at 16000g for 10min. Discard the supernatant and rinse the pellet with 70% cold ethanol.
Air-dry the pellet. Dissolve in TE buffer.
DNA extraction 2: Serapure Beads
DNA extraction 2: Serapure Beads
2h
2h
Alternatively, the DNA extraction can be down with Serapure Beads method, this results in better recovery of genomic DNA in higher N50, which would benefit ONT sequencing.
Take the tube from step 37, place the tube on a magnetic stand, remove all the solution.
Add 600ul pre-warmed Digestion Buffer, plus 4.0ul 10mg/ml RNase A. Mix thoroughly immediately by pipetting up and down 10 times with a wide-bore tip.
Add 10.0ul 20mg/ml Proteinase K. Incubate the tube at 55°C water bath overnight.
Add 200μl (or 1/3 of the lysis buffer volume) of 5M potassium acetate and mix by inverting the tube 20 times in order to obtain a homogenous solution to fully precipitate the proteins and the polysaccharides that will complex with SDS. It is important to incubate at 4°C after the addition of potassium acetate.
Centrifuge at 5000g for 10 minutes at 4°C. Transfer the supernatant to a new 1.5 ml tube without disturbing the pellet.
Add one volume of Bead Binding Buffer and 1:18 (v:v) of Serapure beads previously prepared (vortex the beads solution for 20 seconds before use to ensure that the beads are completely resuspended).
Mix by inverting the tube 20 times. Incubate with a gentle mixer for 10 minutes at room temperature.
Quick spin the tube to remove the liquid from the cap. Place the tube in a magnetic stand until the solution becomes clear (2-3min). The actual time required to collect beads may vary according to samples.
Remove the supernatant without disturbing the beads pellet. Add 1 ml of Bead Wash Buffer, remove the tube from the magnetic rack and mix by inverting the tube 20 times.
Quick spin the tube to remove the liquid from the cap. Place the tube in a magnetic stand until the solution becomes clear (2-3min).
Repeat step 53-54.
Quick spin the tube to remove the liquid from the cap. Place the tube in a magnetic stand to remove the remaining Bead Wash Buffer.
Let the beads air-dry for 1 minute with the cap open. Do not let the beads dry more than 1 minute as this will significantly decrease elution efficiency.
Add 80 μl of TE buffer preheated to 50°C.
Resuspend the beads by flicking the tube. It is important that the beads are not aggregated.
Quick spin the tube. Place the tube in the magnetic rack. Let the solution to become clear. If DNA solution is highly concentrated, it can take a long time. In this case, it is recommended to let the tube in the magnetic rack overnight or to add more elution buffer.
Transfer 75 μl of the eluted gDNA solution in a new tube.
ONT library prepartion and sequencing
ONT library prepartion and sequencing
3d
3d
Follow Oxford Nanopore protocol of LSK109 for adaptor ligation.
Load 500ng of the ligated DNA to R9.4.1 flow cell. Use the Flow Cell Wash Kit to wash the flow cell.
Reload the flow cell every 24h.