TITLE: BIND&amp;MODIFY: Long-range single-molecule mapping of chromatin modification in eukaryotes. V.2
AUTHORS: 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

[DESCRIPTION]
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.

[STEPS]
SECTION: OVERVIEW
1. 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
dx.doi.org/10.17504/protocols.io.b2ahqab6

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

  

SECTION: REAGENT SETUP
2. 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. 
 
    ABC1ReagentQuantityFinal Concentration21M HEPES pH 7.9200ul20mM31M KCl100ul10mM41M CaCl210ul1mM51M MnCl210ul1mM6H2O9750ul 7Total10ml 


Wash Buffer: Prepare fresh, and store the buffer at 4°C up to 1 week.

    ABC1ReagentQuantityFinal Concentration21M HEPES pH 7.51.0ml20mM35M NaCl1.5ml150mM42M Spermidine12.5ul0.5mM5Roche Proteinase Inhibitor cocktail tablet1 tablet1X6H2O47.5ml 7Total50ml 


Dig-wash Buffer: Prepare fresh, store the buffer at 4°C up to 2 days.

    ABC1ReagentQuantityFinal Concentration25% Digitonin400ul0.05%31X Wash Buffer39.6ml 4Total 40ml 


Tween-wash Buffer: Prepare fresh, store the buffer at 4°C up to 2 days.

    ABC1ReagentQuantityFinal Concentration25% Digitonin400ul0.05%31X Wash Buffer39.6ml 4Total 40ml 


Methylation Buffer: Prepare fresh. Add SAM only before the methylation reaction.

    ABC1ReagentQuantityFinal Concentration210X CutSmart Buffer30ul1x350X Roche Proteinase Inhibitor cocktail6ul1x45% Digitonin3ul0.05%532mM SAM7.5ul800uM620% BSA1.5ul0.10%7H2O252ul 8Total300ul 


Digestion Buffer: Prepare fresh.
    ABC1ReagentQuantityFinal Concentration2Polyvinylpyrrolidone 40 0.1g1%3Sodium metabisulfite0.1g1%45M NaCl1.0ml0.5M51M Tris-HCl, pH 8.01.0ml0.5M60.5M EDTA1.0ml50mM720% SDS625ul1.25%8H2O6375ul 9Total10ml 


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. 
    ABC1ReagentQuantityFinal Concentration250% PEG80003.6 ml18%35M NaCl2 ml1M41M Tris-HCl, pH 8.00.1ml10mM50.5M EDTA20ul1mM6100% tween 205ul0.05%7H2O4275ul 8Total10ml 


Bead Binding Buffer: Store the solution at 4°C for 1 week.
    ABC1ReagentQuantityFinal Concentration250% PEG 80004ml20%35M NaCl6ml3.0M4Total10ml 
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.  
    ABC1ReagentQuantityFinal Concentration2Absolute Ethanol35ml70%3H2O15ml 4Total50ml 


 
 
 
SECTION: Cells Preparation
3. Harvest fresh cells at room temperature.


Note: Use Eppendorf DNA LowBind tube during the whole protocol to reduce cell/DNA loss.
SECTION: Bind cells or nuclei to Concanavalin A-coated beads
11. 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. 
SECTION: Primary Antibody Binding
21. Quick spin the tube to remove the liquid from the cap. Place the tube on a magnetic stand to clear, remove the liquid.
SECTION: Protein A Block
18. Quick spin the tube to remove the liquid from the cap. Place the tube on a magnetic stand to clear, remove the liquid.
SECTION: pA-M.EcoGII Binding
28. Add 10ul pA-M.EcoGII recombinant enzyme into 190ul Tween-wash Buffer, mix gently by pipetting. 
SECTION: Cells Preparation
4. Centrifuge cells at 300g for 5 min at 4°C.
SECTION: Cells Preparation
5. Resuspend the cells in 1ml cold PBS, repeat step 5-step 6 twice.
SECTION: Cells Preparation
6. 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. 
SECTION: Cells Preparation
7. Add freshly prepared 1% formaldehyde into the resuspended cells (100ul into 900ul cells), and put at room temperature for 10 min. 
SECTION: Cells Preparation
8. Stop the crosslinking by adding 1.25M glycine to twice molar ratio of formaldehyde (60ul into 1ml cell fixing reaction). 
SECTION: Cells Preparation
9. 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.  
SECTION: Cells Preparation
10. 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. 
SECTION: Bind cells or nuclei to Concanavalin A-coated beads
12. 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).
SECTION: Bind cells or nuclei to Concanavalin A-coated beads
13. 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.
SECTION: Bind cells or nuclei to Concanavalin A-coated beads
14. 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. 
SECTION: Bind cells or nuclei to Concanavalin A-coated beads
15. 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.
SECTION: Primary Antibody Binding
22. 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. 
SECTION: Primary Antibody Binding
23. 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.
SECTION: Protein A Block
19. Resuspend the cells in 200ul ice cold Tween-wash Buffer with gentle vortexing. 
SECTION: Methyltransferase Activation
35. Quick spin the tube from step 34. Place the tube on a magnetic stand, carefully remove the solution. 


SECTION: Protein A Block
20. Add 2.5ul 50ug/ul protein A.  Place on end-over-end rotator at room temperature for 10min.
SECTION: Primary Antibody Binding
24. Place the tube on end-over-end rotator at 4°C for 2h.
SECTION: Primary Antibody Binding
25. Quick spin the cellls with primary antibody. Place the cells with primary antibody on a magnetic stand, carefully remove the solution. 
SECTION: Primary Antibody Binding
26. Add 1ml Tween-wash Buffer. Invert the tube 10 times to resuspend the beads.
SECTION: Primary Antibody Binding
27. Repeat step 23-25 twice.
SECTION: pA-M.EcoGII Binding
29. Quick spin the tube from step 28. Place the tube on a magnetic stand, carefully remove the solution. 
SECTION: pA-M.EcoGII Binding
30. Add the pA-M.EcoGII containing buffer to the cells with gentle vortexing. 
SECTION: pA-M.EcoGII Binding
31. Place the tube on end-over-end rotator at room temperature for 1h.
SECTION: pA-M.EcoGII Binding
32. Quick spin the cells with pA-M.EcoGII. Place the cells with pA-M.EcoGII on a magnetic stand, carefully remove the solution. 
SECTION: pA-M.EcoGII Binding
33. Add 1ml Tween-wash Buffer. Invert the tube 10 times to resuspend the beads.
SECTION: pA-M.EcoGII Binding
34. Repeat step 30-31 twice.
SECTION: Methyltransferase Activation
36. Add 300ul Methylation Buffer. Invert the tube 10 times to resuspend the beads.
SECTION: Methyltransferase Activation
37. 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.
SECTION: DNA extraction 1:PCI
38. 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.
SECTION: DNA extraction 2: Serapure Beads
46. 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.
SECTION: DNA extraction 1:PCI
39. 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. 
SECTION: DNA extraction 1:PCI
40. Add 300ul PCI and vortexing at highest speed for 30s. Invert the tube 10 times to mix thoroughly. Centrifuge at 16000g for 5min.
SECTION: DNA extraction 1:PCI
41. 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.
SECTION: DNA extraction 1:PCI
42. Transfer the upper liquid aqueous phase to a new 1.5ml centrifuge tube. Add 1/10 volume of 3M sodium acetate solution.  
SECTION: DNA extraction 1:PCI
43. Add 2.5x volume of ice cold 100% ethanol in the solution to precipitate DNA. Incubate the tube at -20 overnight.
SECTION: DNA extraction 1:PCI
44. Centrifurge at 16000g for 10min. Discard the supernatant and rinse the pellet with 70% cold ethanol.
SECTION: DNA extraction 1:PCI
45. Air-dry the pellet. Dissolve in TE buffer.
SECTION: DNA extraction 2: Serapure Beads
47. Take the tube from step 37, place the tube on a magnetic stand, remove all the solution.
SECTION: DNA extraction 2: Serapure Beads
48. 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. 
SECTION: DNA extraction 2: Serapure Beads
49. Add 10.0ul 20mg/ml Proteinase K. Incubate the tube at 55°C water bath overnight. 
SECTION: DNA extraction 2: Serapure Beads
50. 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.
SECTION: DNA extraction 2: Serapure Beads
51. Centrifuge at 5000g for 10 minutes at 4°C. Transfer the supernatant to a new 1.5 ml tube without disturbing the pellet.
SECTION: DNA extraction 2: Serapure Beads
52. 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).
SECTION: DNA extraction 2: Serapure Beads
53. Mix by inverting the tube 20 times. Incubate with a gentle mixer for 10 minutes at room temperature.
SECTION: DNA extraction 2: Serapure Beads
54. 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.
SECTION: DNA extraction 2: Serapure Beads
55. 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.
SECTION: DNA extraction 2: Serapure Beads
56. 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). 
SECTION: DNA extraction 2: Serapure Beads
57. Repeat step 53-54.
SECTION: DNA extraction 2: Serapure Beads
58. 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.
SECTION: DNA extraction 2: Serapure Beads
59. 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.
SECTION: DNA extraction 2: Serapure Beads
60. Add 80 μl of TE buffer preheated to 50°C.
SECTION: DNA extraction 2: Serapure Beads
61. Resuspend the beads by flicking the tube. It is important that the beads are not aggregated.
SECTION: DNA extraction 2: Serapure Beads
62. 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.
SECTION: DNA extraction 2: Serapure Beads
63. Transfer 75 μl of the eluted gDNA solution in a new tube.
SECTION: ONT library prepartion and sequencing
64. Follow Oxford Nanopore protocol of LSK109 for adaptor ligation.
SECTION: ONT library prepartion and sequencing
65. Load 500ng of the ligated DNA to R9.4.1 flow cell. Use the Flow Cell Wash Kit to wash the flow cell.
SECTION: ONT library prepartion and sequencing
66. Reload the flow cell every 24h. 
SECTION: Bind cells or nuclei to Concanavalin A-coated beads
16. 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.  
SECTION: Bind cells or nuclei to Concanavalin A-coated beads
17. 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.