TITLE: in situ Hi-C
AUTHORS: Florian  Noack, Jeisimhan Diwakar, Boyan Bonev

[DESCRIPTION]
Protocol was adapted from the HiC 2.0 protocol (Belaghzal et al.; 2018).

[STEPS]
SECTION: Cell fixation
1. 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 RT with slow rotation.  
SECTION: Cell fixation
2. 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
3. Centrifuge for 5 minutes at 500xg at 4°C. Discard supernatant into an appropriate collection container.
SECTION: Cell fixation
4. 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 either stained for ImmunoFACS, directly used for in-situ HiC or pelleted (2500xg for 5 minutes at 4˚C) and then snap frozen in liquid nitrogen for storage at -80˚C.
SECTION: in situ Hi-C: Cell Lysis
5. NOTE: this protocol was optimized for ~3x104 cells. 

Pellet fixed cells for 5 minutes at 2500xg (4˚C) and carefully resuspend them with 100µl of ice-cold Lysis Buffer.

If cells were frozen, thaw the cell pellet first on ice. 
SECTION: in situ Hi-C: Cell Lysis
6. Incubate on ice for exactly 10 minutes and centrifuge at 2500xg for 5 minutes  at 4°C. and remove the supernatant.   
SECTION: in situ Hi-C: Cell Lysis
7. Wash nuclei once with 190µl wash buffer. Centrifuge at 2500xg for 5 minutes and remove as much of the supernatant as possible without disturbing the nuclei pellet. 
SECTION: in situ Hi-C: DpnII Digestion and biotin fill-in
8. Gently resuspend nuclei pellet in 20μl of 0.5% SDS (Invitrogen, Cat. N.: AM9823) and incubate at 62°C for 10 minutes. Afterwards place on room temperature.

SECTION: in situ Hi-C: DpnII Digestion and biotin fill-in
9. Add first 56μl of water and then 10μl 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: in situ Hi-C: DpnII Digestion and biotin fill-in
10. Add 10μl of 10X DpnII buffer (New England Biolabs, Cat. N.: R0543) and 4µl (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: in situ Hi-C: DpnII Digestion and biotin fill-in
11. Next day incubate samples at 62°C for 20 minutes, then cool the samples to room temperature.
SECTION: in situ Hi-C: DpnII Digestion and biotin fill-in
12. To fill in the restriction overhangs and mark the DNA ends with biotin, add 20μl 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 min).
SECTION: in situ Hi-C: Proximity ligation and decrosslinking
13. Add 360μl of the Ligation master mix. Mix by inverting and incubate at least for 6h at 16°C (900 rpm mixing, 10 sec every 30 min). Alternativly the ligation reaction can be also performed overnight. 
SECTION: in situ Hi-C: Proximity ligation and decrosslinking
14. Degrade proteins by adding 5μl of 20mg/ml Proteinase K (New England Biolabs, Cat. N.: P8107) and 48μl of 10% SDS (Invitrogen, Cat. N.: AM9823) and incubate at 55°C for 30 minutes.
SECTION: in situ Hi-C: Proximity ligation and decrosslinking
15. Add 53μl of 5M NaCl (ThermoFisher, Cat. N.: AM9760G) and incubate at 68°C overnight (900 rpm 10sec, every 5 min.).
SECTION: in situ Hi-C: Proximity ligation and decrosslinking
16. Cool tubes at room temperature, add 1:1 volume Phenol/Chloroform/Isoamylalcohol (Invitrogen, Cat. N.: 15593031), vortex for at least 30 seconds and spin with maximal speed for 10 minutes at 4˚C.
SECTION: in situ Hi-C: Proximity ligation and decrosslinking
17. 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: in situ Hi-C: Proximity ligation and decrosslinking
18. Centrifuge at maximal speed for 20 minutes at 4°C. Carefully remove the supernatant by pipetting.
SECTION: in situ Hi-C: Proximity ligation and decrosslinking
19. Wash with 800μl freshly prepared 70% cold ethanol followed by centrifuge at maximal speed for 5 minutes at 4°C. 

SECTION: in situ Hi-C: Proximity ligation and decrosslinking
20. Wash with 400µl freshly prepared 70% cold ethanol and transfer everything to a 0.5 ml tube (make sure the glycogen pellet is transfered). Centrifuge at maximal speed for 5 minutes at 4°C. 
SECTION: in situ Hi-C: Proximity ligation and decrosslinking
21. Remove as much as possible of the ethanol and air-dry the pellet. Dissolve pellet in 17µl of 10mM Tris-buffer (pH 7.5) (ThermoFisher, Cat. N.: 15567027) and incubate at 37°C for 15 minutes to fully dissolve DNA.
SECTION: in situ Hi-C: DNA shearing (Covaris)
22. Transfer 17µl 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: in situ Hi-C: DNA shearing (Covaris)
23. Quantify the sheared DNA using the Qubit dsDNA High Sensitivity Assay. 
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
27. Wash the beads once with 100µl 1x (!!!) NEB T4 DNA ligase buffer (New England Biolabs, Cat. N.: B0202S).
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
30. Wash the beads once with 100µl 1x (!!!) NEB2 Buffer (New England Biolabs, Cat. N.: B7002S).


SECTION: in situ Hi-C: adapter ligation and library amplification
33. Wash the beads once with 100μl 1x (!!!) Quick ligation reaction buffer (New England Biolabs, Cat. N.: B6058). 
SECTION: in situ Hi-C: adapter ligation and library amplification
34. Resuspend in 50μl of 1x (!!!) Quick ligation reaction buffer (New England Biolabs, Cat. N.: B6058), add 2µl of sample specific NextFlex index adapters (PerkinElmer, Cat. N.: NOVA-514101) and 2μl of NEB DNA Quick ligase (New England Biolabs, Cat. N.: M2200). Mix thoroughly and incubate for 15 minutes at room temperature. 
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
24. Prepare streptavidin beads by washing 100µl of 10mg/ml MyOne Streptavidin T1 beads (ThermoFisher, Cat. N.: 65602) with 400µl 1x Tween washing buffer. Afterwards resuspend beads in 200µl of 2x Binding buffer.
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
25. Resuspend 15µl of the sheared sample DNA with 185µl nuclease free water and 200µl of the washed MyOne Streptavidin T1 beads in binding buffer. Incubate at room temperature for 15 - 30 minutes with rotation to bind biotinylated DNA to the streptavidin beads.  
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
26. Separate on a magnet and discard the solution. Wash the beads twice by adding 600μl of tween washing buffer, incubating for 2 minutes at 55˚C with mixing (1000 RPM) followed by reclaim the beads using a magnet and discarding the supernatant.
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
28. To repair ends of the sheared DNA and to remove the biotin from unligated ends, resuspend beads in 100μl of Biotin Removal Master Mix and incubate for 30 minutes at room temperature. 
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
29. Separate on a magnet and discard the solution. Wash the beads twice by adding 600μl of tween washing buffer, incubating for 2 minutes at 55˚C with mixing (1000 RPM) followed by reclaim the beads using a magnet and discarding the supernatant.
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
31. Resuspend beads in 100μl of dATP attachment master mix and incubate for 30 minutes at 37°C.
SECTION: in situ Hi-C: Biotin pulldown, on-bead Biotin removal and End-repair
32. Separate on a magnet and discard the solution. Wash the beads twice by adding 600μl of tween washing buffer, incubating for 2 minutes at 55˚C with mixing (1000 RPM) followed by reclaim the beads using a magnet and discarding the supernatant.
SECTION: in situ Hi-C: adapter ligation and library amplification
35. Separate on a magnet and discard the solution. Wash the beads twice by adding 600μl of tween washing buffer, incubating for 2 minutes at 55˚C with mixing (1000 RPM) followed by reclaim the beads using a magnet and discarding the supernatant.
SECTION: in situ Hi-C: adapter ligation and library amplification
36. Wash the beads once with 100µl 10mM Tris -uffer pH 7.5 (ThermoFisher, Cat. N.: 15567027).
SECTION: in situ Hi-C: adapter ligation and library amplification
38. Amplification of the HiC library is performed directly on the beads.

Mix 45µl of the end-repaired DNA with 5µl of NEXTFlex Primer mix (PerkinElmer, Cat. N.: NOVA-514101) and 50µl NEBNext Ultra Q5 II Master Mix (New England Biolabs, Cat. N.: M0544S) to reach a total volume of 100µl. Split the reaction into 5 PCR tubes of 20µl, in order to increase the complexity of the library. 

PCR is performed using following program: 98˚C 30s; {98˚C 10s, 65˚C 75s} x8-10; 65˚C 5 min.; Hold at 10˚C
SECTION: in situ Hi-C: adapter ligation and library amplification
37. Resuspend beads in 42μl 10mM Tris-buffer pH 7.5 (ThermoFisher, Cat. N.: 15567027) to reach a total volume of ~45µl.  
SECTION: in situ Hi-C: adapter ligation and library amplification
39. After amplification, beads are separated using a magentic rack and the supernatant containing the final library is pooled together for each sample (volume ~100µl).

SECTION: in situ Hi-C: adapter ligation and library amplification
40. Final library is purified by adding 70µl of AmpureXP beads (0.7X, Agencourt, Cat. N.: A63881) to the samples followed by a incubation of 10 minutes at room temperature under slow rotation.  
SECTION: in situ Hi-C: adapter ligation and library amplification
42. 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: in situ Hi-C: adapter ligation and library amplification
41. Pellet the beads on a magnatic-rack, remove the supernatant and wash the beads twice with freshly prepared 80% Ethanol. 
SECTION: in situ Hi-C: adapter ligation and library amplification
43. Resuspend the beads with 12µl 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. Quantify the yield using the Qubit dsDNA High Sensitivity Assay (ThermoFisher, Cat. N.. Q32851).