TITLE: MethylHiC 
AUTHORS: Florian  Noack, Boyan  Bonev

[DESCRIPTION]
Protocol was adapted from current protocols (Lee et al.; 2019; Li et al.; 2019) with major modifications mainly at the library preparation step.

[STEPS]
SECTION: Prepare bisulfite conversion control
1. 
NOTE: Control DNA has to prepared only once and can be reused. 

To prepare methylation controls, mix 8µl of CpG methylated pUC19 DNA (Zymo Research, Cat. N.: D5017) with 8µl of unmethylated lambda DNA (Promega, Cat. N.: D1521) in Covaris microTUBE-15 AFA Beads Screw-Cap tubes (Covaris, Cat. N.: 520145).
SECTION: Prepare bisulfite conversion control
2. Shear DNA to a targeted size of 550bp using following parameter on a Covaris S220 sonicator:
Peak Incident Power (W)     18
Duty Factor                             20%
Cycles per Burst                    50
Treatment Time (s)               22
SECTION: Prepare bisulfite conversion control
3. Perform biotin fill-in reaction by mixing:
15µl sheared DNA
75µl steril water 
10µl 10x DpnII buffer (New England Biolabs, Cat. N.: R0543)
20µl Biotin fill-in master mix (see Materials)

Incubate at 23°C for 4 hours (900 RPM mixing; 10 sec. every 5 minutes).
SECTION: Prepare bisulfite conversion control
5. Pellet the beads on a magnatic-rack, remove the supernatant and wash the beads twice with freshly prepared 80% Ethanol. 
SECTION: Cell fixation
9. Resuspend dissociated cells in PBS to reach a maximal cell concentration of 2x106 cells/ml and add freshly prepared 2% Formaldehyde solution to reach a final concentration of 1%. Incubate for 10 minutes at room temperature with slow rotation.  
SECTION: Cell fixation
10. Add 2.0M glycine solution (Invitrogen, Cat. N.: 15527013) to a final concentration of 0.2M to quench the reaction. Incubate at room temperature for 5 minutes with slow rotation.  
SECTION: Cell fixation
11. Centrifuge for 5 minutes at 500xg at 4°C. Discard supernatant into an appropriate collection container.
SECTION: Cell fixation
12. Resuspend cells in 1ml of cold 1x PBS with 0.5% BSA (ThermoFisher, Cat. N.: AM2618) and count the cell number. 

Fixed cells can be pelleted (2500xg for 5 minutes at 4˚C) and either snap frozen in liquid nitrogen for storage at -80˚C or directly used for MethylHiC. 
SECTION: MethylHiC: Cell Lysis
13. NOTE: this protocol was optimized for 2x105 cells. 

If cells were frozen, thaw the cell pellet on ice. Carefully resuspend cells with 200µl of ice-cold MethylHiC Lysis Buffer. 
SECTION: MethylHiC: Cell Lysis
14. Incubate on ice for exactly 10 minutes and centrifuge at 2500xg for 5 minutes  at 4°C. and remove the supernatant.  
SECTION: MethylHiC: DpnII Digestion and biotin fill-in
16. Gently resuspend nuclei pellet in 40μl of 0.5% SDS (Invitrogen, Cat. N.: AM9823) and incubate at 62°C for 10 minutes. Afterwards place at room temperature.

SECTION: MethylHiC: DpnII Digestion and biotin fill-in
17. Add first 112μl of water and then 20μl of freshly made 10% Triton X-100 solution (Sigma Aldrich, Cat. N.: X100) to quench the SDS. Mix well by carefully pipetting up/down  (avoiding excessive foaming). Incubate at 37°C for 15 minutes on a thermomixer with a rotation speed of 600 rpm.
SECTION: MethylHiC: DpnII Digestion and biotin fill-in
18. Add 20μl of 10X DpnII buffer (New England Biolabs, Cat. N.: R0543) and 8µl (400U) of DpnII restriction enzyme (New England Biolabs, Cat. N.: R0543) and digest chromatin overnight at 37°C with a rotation speed of 600 rpm.
SECTION: MethylHiC: DpnII Digestion and biotin fill-in
19. Next day incubate samples at 62°C for 20 minutes, then cool the samples to room temperature.
SECTION: MethylHiC: DpnII Digestion and biotin fill-in
20. To fill in the restriction overhangs and mark the DNA ends with biotin, add 40μl of Biotin fill-in master mix . Mix by pipetting carefully and incubate for 4 hours at 23°C (900 rpm mixing; 10 sec. every 5 minutes).
SECTION: MethylHiC: Proximity ligation and decrosslinking
21. Add 720μl of the Ligation master mix. Mix by inverting and incubate at least for 6 hours at 16°C (900 rpm mixing, 10 sec. every 30 min.). Alternatively, the ligation reaction can also be performed overnight. 
SECTION: MethylHiC: Proximity ligation and decrosslinking
22. Degrade proteins by adding 10μl of 20mg/ml Proteinase K (New England Biolabs, Cat. N.: P8107) and 96μl of 10% SDS (Invitrogen, Cat. N.: AM9823) and incubate at 55°C for 30 minutes.
SECTION: MethylHiC: Proximity ligation and decrosslinking
23. Add 106μl of 5M NaCl (ThermoFisher, Cat. N.: AM9760G) and incubate at 68°C overnight (900 rpm, 10 sec., every 5 min.).
SECTION: MethylHiC: Proximity ligation and decrosslinking
24. Cool tubes at room temperature. Split reaction into 2x 2ml tubes and add 1:1 volume Phenol/Chloroform/Isoamylalcohol (Invitrogen, Cat. N.: 15593031), vortex for at least 30seconds and spin with maximal speed for 10 minutes at 4˚C.
SECTION: MethylHiC: Proximity ligation and decrosslinking
25. Take the upper aqueous phase into a 2ml tube and resuspend with 2x volumes of ice cold 100% ethanol, 1/10th of the volume 3M NaAc (Ambion, Cat. N.: AM9740) and 1µl Glycogen (ThermoFisher, Cat. N.: 10814010). Store the mix at -20˚C at least for 1 hour or -80˚C for 15 minutes.
SECTION: MethylHiC: Proximity ligation and decrosslinking
26. Centrifuge at maximal speed for 20 minutes at 4°C. Carefully remove the supernatant by pipetting.
SECTION: MethylHiC: Proximity ligation and decrosslinking
27. Wash with 800μl freshly prepared 70% cold ethanol followed by centrifuge at maximal speed for 5 minutes at 4°C. 

SECTION: MethylHiC: Proximity ligation and decrosslinking
28. Wash each tube with 200µl freshly prepared 70% cold Ethanol and transfer the total 400µl to a 0.5ml tube (make sure glycogen pellet is transferred). Centrifuge at maximal speed for 5 minutes at 4°C. 
SECTION: MethylHiC: Proximity ligation and decrosslinking
29. Remove as much as possible of the ethanol and airdry the pellet. Dissolve pellet in 16ul of 10mM Tris buffer pH 7.5 (ThermoFisher, Cat. N.: 15567027) and incubate at 37°C for 15 minutes to fully dissolve DNA.
SECTION: MethylHiC: DNA shearing (Covaris)
30. Transfer 16µl of the sample into a Covaris microTUBE-15 AFA Beads Screw-Cap tube (Covaris, Cat. N.: 520145) and shear DNA to a target size of 550bp using following parameter on a Covaris S220 sonicator:
Peak Incident Power (W)     18
Duty Factor                             20%
Cycles per Burst                    50
Treatment Time (s)               22
SECTION: MethylHiC: DNA shearing (Covaris)
31. Quantify the yield using the Qubit dsDNA High Sensitivity Assay. 
SECTION: MethylHiC: Biotin removal
32. 
Mix 15µl of the sheared sample with 5µl of the Endrepair master mix.
SECTION: MethylHiC: Biotin removal
33. Incubate for 4 hours at 20˚C followed by a heat inactivation for 20 minutes at 75˚C. Sample can be stored at -20˚C. 


SECTION: MethylHiC: Bisulfite Conversion
34. NOTE: for the highest yield the libraries should be prepared directly after the bisulfite conversion. 

Prior the bisulfite conversion add roughly 0.05% sheared-biotinylated control DNA to the sample. Proceed with the bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manual instructions until the elution step.   
SECTION: MethylHiC: Bisulfite Conversion
35. Elute the bisulfite converted DNA in either 16µl (<100ng DNA input) or 31µl (>100ng DNA input) low EDTA TE water provided with the Accel-NGS® Methyl-Seq DNA Library Kit (Swift Bioscience, Cat. N.: 30024). 
SECTION: MethylHiC: Library construction and Biotin pulldown
37. 
Per sample prepare 25µl Dynabeads MyOne Streptavidin T1 beads (ThermoFisher, Cat. N.: 65602) by washing them with 400µl 1x Tween washing buffer and resuspend the beads in 60µl of 2x Binding buffer.
SECTION: MethylHiC: Library construction and Biotin pulldown
39. Wash beads 4 times with 400µl 1x Tween washing buffer for 2 minutes at 55˚C.
SECTION: MethylHiC: Library construction and Biotin pulldown
40. After the last wash remove as much as possible from the Tween washing buffer and wash beads twice with 400µl nuclease free water. Thereafter resuspend beads in 20µl nuclease free water. 
SECTION: MethylHiC: Library construction and Biotin pulldown
41. Mix 20µl of the streptavidin bound sample with 30µl Library amplification mix. Amplify library using the following PCR program: 95˚C 30s; {95˚C 15s, 61˚C 30s, 68 ˚C 60s}x8-14; 68˚C 5min; Hold at 10˚C.
SECTION: MethylHiC: Library construction and Biotin pulldown
42. After final library amplification separate the streptavidin beads using a magnetic rack and transfer the supernatant into a new tube. Perform a 0.6x AmpureXP (Agencourt, Cat. N.: A63881) purification by mix 30µl of room temperature AmpureXP beads to the samples followed by an incubation of 10 minutes at room temperature under slow rotation.  
SECTION: Prepare bisulfite conversion control
8. Quantify the yield using the Qubit dsDNA High Sensitivity Assay (ThermoFisher, Cat. N.: Q32851). Low DNA amounts expected. Control DNA can be stored at -20˚C. 

SECTION: MethylHiC: Cell Lysis
15. Wash nuclei once with 190µl MethylHiC wash buffer. Centrifuge at 2500xg for 5 minutes and remove as much of the supernatant as possible without disturbing the nuclei pellet. 
SECTION: MethylHiC: Library construction and Biotin pulldown
36. For <100ng pre-bisulfite DNA input use 15µl of bisulfite converted DNA for the Accel-NGS® Methyl-Seq DNA Library Kit according to the manual instruction until the ligation step (step 16: 15 minutes at 25˚C). 

For >100ng pre-bisulfite DNA input use 30µl of bisulfite converted DNA for the Accel-NGS® Methyl-Seq DNA Library Kit with the double reaction volume according to the manual instruction until the ligation step (step 16: 15 minutes at 25˚C). 
SECTION: MethylHiC: Library construction and Biotin pulldown
38. Add nuclease free water to the ligation reaction to reach a final volume of 60µl and subsequently add 60µl of the washed MyOne Streptavidin T1 beads in 2x Binding buffer. Incubate for 15 minutes at room temperature under slow rotation.  
SECTION: Prepare bisulfite conversion control
4. Perform a 1x AmpureXP (Agencourt, Cat. N.: A63881) purification by adding 120 µl of room temperature AmpureXP beads to the samples followed by a incubation of 10 minutes at room temperature under slow rotation.  
SECTION: Prepare bisulfite conversion control
6. After the last wash remove as much as possible of the 80% Ethanol and air-dry the beads for approximately 5 minutes at room temperature. 
SECTION: Prepare bisulfite conversion control
7. Resuspend the beads with 50µl nuclease-free water, incubate for 10 minutes at room temperature, pellet the beads on a magnetic-rack and transfer the DNA containing supernatant into a new tube.  
SECTION: MethylHiC: Library construction and Biotin pulldown
43. Pellet the beads on a magnetic-rack, remove the supernatant and wash the beads twice with freshly prepared 80% Ethanol. 
SECTION: MethylHiC: Library construction and Biotin pulldown
44. After the last wash remove as much as possible of the 80% Ethanol and airdry the beads for approximatly 5 minutes at room temperature. 
SECTION: MethylHiC: Library construction and Biotin pulldown
45. Resuspend the beads with 10µl nuclease-free water, incubate for 10 minutes at room temperature, pellet the beads on a magnetic-rack and transfer the DNA containing supernatant into a new tube.  
SECTION: MethylHiC: Library construction and Biotin pulldown
46. Quantify the yield using the Qubit dsDNA High Sensitivity Assay (ThermoFisher, Cat. N.: Q32851).