TITLE: BIND&amp;MODIFY: Long-range single-molecule mapping of chromatin modification in eukaryotes
AUTHORS: Zhe Weng, Fengying Ruan, Weitian Chen, Zhe Xie, Yeming Xie, Chen Zhang, Zhichao Chen, Juan Wang, Yuxin Sun, Yitong Fang, Mei Guo, Yiqin Tong, Yaning Li, Chong Tang

[DESCRIPTION]
Here we describe a powerful method, BIND&MODIFY, for probing histone modifications and transcription factors at single molecular level. 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: 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 5x105 cells. The following is for 4 samples. 
SECTION: Bind primary antibody
16. Quick spin the tube to remove the liquid from the cap. Place the tube on a magnetic stand to clear, remove the liquid.
SECTION: Bind pA-M.EcoGII  recombinant enzyme
23. Add 20ul pA-M.EcoGII recombinant enzyme into 130ul dig-wash Buffer, mix gently by pipetting. 
SECTION: Methyltransferase activation
30. Quick spin the tube from step 29. Place the tube on a magnetic stand, carefully remove the solution. 
SECTION: DNA extraction: Serapure Beads
41. Take the tube from step 32, place the tube on a magnetic stand, remove all the solution.
SECTION: DNA extraction:PCI
33. Take the tube from step 32. 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: 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. 
   ReagentQuantityFinal Concentration1M HEPES pH 7.9200ul20mM1M KCl100ul10mM1M CaCl210ul1mM1M MnCl210ul1mMH2O9750ul Total10ml 


Wash Buffer: Prepare fresh, and store the buffer at 4°C up to 1 week.
   ReagentQuantityFinal Concentration1M HEPES pH 7.51.0ml20mM5M NaCl1.5ml150mM2M Spermidine12.5ul0.5mMRoche Proteinase Inhibitor cocktail tablet1 tablet1XH2O47.5ml Total50ml 
 

Dig-wash Buffer: Prepare fresh, store the buffer at 4°C up to 2 days.
   ReagentQuantityFinal Concentration5% Digitonin400ul0.05%1X Wash Buffer39.6ml Total 40ml 
 

Antibody Buffer: Prepare fresh.
   ReagentQuantityFinal Concentration1M HEPES pH 7.5100ul20mM5M NaCl150ul150mM2M Spermidine1.25ul0.5mM50X Roche Proteinase Inhibitor cocktail100ul1X5% Digitonin50ul0.05%0.5M EDTA20ul2mM20% BSA25ul0.10%H2O4555ul Total5ml 
 

Methylation Buffer: Prepare fresh, store the buffer at 4°C up to 2 days.
   ReagentQuantityFinal Concentration10X CutSmart Buffer30ul1x50X Roche Proteinase Inhibitor cocktail6ul1x5% Digitonin3ul0.05%32mM SAM7.5ul800uM20% BSA1.5ul0.10%H2O252ul Total300ul 
 

Digestion Buffer: Prepare fresh.
   ReagentQuantityFinal ConcentrationPolyvinylpyrrolidone 40 0.1g1%Sodium metabisulfite0.1g1%5M NaCl1.0ml0.5M1M Tris-HCl, pH 8.01.0ml0.5M0.5M EDTA1.0ml50mM20% SDS625ul1.25%H2O6375ul Total10ml 
 
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.
   ReagentQuantityFinal ConcentrationSerapure Beads 20ul2%PEG80001.8ml18%5M NaCl2ml1M1M Tris-HCl, pH 8.00.1ml10mM0.5M EDTA20ul1mMH2O6060ul Total10ml 
 


Bead Binding Buffer: Store the solution at 4°C for 1 week.
   ReagentQuantityFinal ConcentrationPEG 80002.0g20%5M NaCl6ml3.0MH2O4ml Total10ml 
 
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.  
   ReagentQuantityFinal ConcentrationAbsolute Ethanol35ml70%H2O15ml Total50ml 
 

SECTION: OVERVIEW
1. This protocol describes step-by-step guidelines for BIND&MODIFY method.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) 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. 

  
  


SECTION: DNA extraction: Serapure Beads
42. 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: 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: 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 8.  

Normally this protocol works for 5x105 cells per methylation reaction. Aliquot 4x106 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: 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: 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 5x105 cells per methylation reaction. Aliquot 4x106 cells per centrifuge tube for 8 tube methylation reaction. 
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 4x106 cells prepared from step 7 or 11. Place on end-over-end rotator for 10min.
SECTION: Bind primary antibody
17. Resuspend the cells in 400ul ice cold Antibody Buffer with gentle vortexing. Divide into 8 1.5ml Eppendorf LowBind tubes, and 50ul each tube. Scale up or down based on specific applications. 
SECTION: Bind primary antibody
18. 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: Bind primary antibody
19. Place the tube on end-over-end rotator at 4°C overnight.
SECTION: Bind primary antibody
20. Quick spin the cellls with primary antibody. Place the cells with primary antibody on a magnetic stand, carefully remove the solution. 
SECTION: Bind pA-M.EcoGII  recombinant enzyme
24. Quick spin the tube from step 22. Place the tube on a magnetic stand, carefully remove the solution. 
SECTION: Bind pA-M.EcoGII  recombinant enzyme
25. Add the pA-M.EcoGII containing buffer to the cells with gentle vortexing. 
SECTION: Bind pA-M.EcoGII  recombinant enzyme
26. Place the tube on end-over-end rotator at room temperature for 1h.
SECTION: Bind pA-M.EcoGII  recombinant enzyme
27. Quick spin the cells with pA-M.EcoGII. Place the cells with pA-M.EcoGII on a magnetic stand, carefully remove the solution. 
SECTION: Bind primary antibody
21. Add 1ml Dig-Wash Buffer. Invert the tube 10 times to resuspend the beads.
SECTION: Bind primary antibody
22. Repeat step 20-21 twice.
SECTION: Bind pA-M.EcoGII  recombinant enzyme
28. Add 1ml Dig-Wash Buffer. Invert the tube 10 times to resuspend the beads.
SECTION: Bind pA-M.EcoGII  recombinant enzyme
29. Repeat step 27-28 twice.
SECTION: Methyltransferase activation
31. Add 300ul Methylation Buffer. Invert the tube 10 times to resuspend the beads.
SECTION: DNA extraction:PCI
34. 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: Methyltransferase activation
32. Incubate at 37°C thermomixer at 300rpm for 30min. Supplement 7.5ul 32mM SAM at 7.5min, 15min, and 22.5min. 
SECTION: ONT library prepartion and sequencing
58. Follow Oxford Nanopore protocol of LSK109 for adaptor ligation.
SECTION: DNA extraction:PCI
35. 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:PCI
36. 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:PCI
37. 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:PCI
38. Add 2.5x volume of ice cold 100% ethanol in the solution to precipitate DNA. Incubate the tube at -20 overnight.
SECTION: DNA extraction:PCI
39. Centrifurge at 16000g for 10min. Discard the supernatant and rinse the pellet with 70% cold ethanol.
SECTION: DNA extraction:PCI
40. Air-dry the pellet. Dissolve in TE buffer.
SECTION: DNA extraction: Serapure Beads
43. Add 10.0ul 20mg/ml Proteinase K. Incubate the tube at 55°C water bath overnight. 
SECTION: DNA extraction: Serapure Beads
44. 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: Serapure Beads
45. 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: Serapure Beads
46. 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: Serapure Beads
47. Mix by inverting the tube 20 times. Incubate with a gentle mixer for 10 minutes at room temperature.
SECTION: DNA extraction: Serapure Beads
48. 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: Serapure Beads
49. 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: Serapure Beads
50. 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: Serapure Beads
51. Repeat step 49-50.
SECTION: DNA extraction: Serapure Beads
52. 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: Serapure Beads
53. 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: Serapure Beads
54. Add 80 μl of TE buffer preheated to 50°C.
SECTION: DNA extraction: Serapure Beads
55. Resuspend the beads by flicking the tube. It is important that the beads are not aggregated.
SECTION: DNA extraction: Serapure Beads
56. 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: Serapure Beads
57. Transfer 75 μl of the eluted gDNA solution in a new tube.
SECTION: ONT library prepartion and sequencing
59. 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
60. Reload the flow cell every 24h.