TITLE: 3DRAM-seq enables joint epigenome profiling of spatial genome organization, chromatin accessibility and DNA methylation at high resolution
AUTHORS: Florian Noack, Boyan Bonev

[DESCRIPTION]
3DRAM-seq (3D genome, RNA, Accessibility and Methylation sequencing) is a novel multiomic method that simultaneously interrogates spatial genome organization, chromatin accessibility, DNA methylation and gene expression at high resolution. It utilized enzymatic treatment of bulk fixed nuclei with the GpC methyltransferase M.CviPI prior to restriction digestion, biotin fill-in and proximity ligation as in Hi-C. The resulting 3C DNA is then sheared to ~550bp, followed by bisulfite conversion and library preparation based on the Accel-NGS® Methyl-Seq DNA but using streptavidin beads after the adaptor ligation step to enrich for fragments containing informative contacts. 3DRAM-seq can be performed with as few as 200 000 cells and can be combined with immunoFACS to profile the epigenome landscape in a cell-type-specific manner. 


 







[STEPS]
SECTION: 3DRAM-seq: Methylation controls
1. NOTE: Control DNA has to prepared only once and can be reused. 

To prepare GpC methylated control DNA, mix 10µl of CpG methylated pUC19 DNA (Zymo Research, Cat. N.: D5017) with 10µl of unmethylated lambda DNA (Promega, Cat. N: D1521). 
SECTION: 3DRAM-seq: Methylation controls
8. Shear the 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: 3DRAM-seq: Methylation controls
9. 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 min.).
SECTION: 3DRAM-seq: Methylation controls
10. 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 RT under slow rotation.  
SECTION: 3DRAM-seq: Methylation controls
11. Pellet the beads on a magnatic-rack, remove the supernatant and wash the beads twice with freshly prepared 80% Ethanol. 
SECTION: 3DRAM-seq: Methylation controls
12. After the last wash remove as much as possible of the 80% Ethanol and air-dry the beads for approximately 5 minutes at RT. 
SECTION: 3DRAM-seq: Methylation controls
13. Resuspend the beads with 50µl nuclease-free water, incubate for 10 minutes at RT, pellet the beads on a magnetic-rack and transfer the DNA containing supernatant into a new tube.  
SECTION: 3DRAM-seq: Methylation controls
14. Quantify the yield using the Qubit dsDNA High Sensitivity Assay (ThermoFisher, Cat. N. Q32851). Control DNA can be stored at -20˚C. 
SECTION: Cell fixation
15. 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 min. at RT with slow rotation.  
SECTION: Cell fixation
16. 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 at rotating wheel.
SECTION: Cell fixation
17. Centrifuge for 5 minutes at 500xG at 4°C. Discard supernatant into an appropriate collection container.
SECTION: Cell fixation
18. Resuspend cells in 1ml of cold 1x PBS with 0.5% BSA (ThermoFisher, Cat. N: AM2618) and count the cell number. 
SECTION: 3DRAM-seq: Cell lysis and GpC methylation
23. NOTE: this protocol was optimized for ~2x105 cells. 

Pellet fixed cells for 5min. at 2500xg (4˚C) and carefully resuspend them with 190µl of ice-cold Lysis Buffer.

If cells were frozen, thaw the cell pellet first on ice. 

SECTION: 3DRAM-seq: Cell lysis and GpC methylation
24. Incubate on ice for exactly 10 minutes and centrifuge at 2500xg for 5 minutes  at 4°C. and remove the supernatant.  
SECTION: 3DRAM-seq: DpnII Digestion and biotin fill-in
28. 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: 3DRAM-seq: DpnII Digestion and biotin fill-in
30. 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 rotationspeed of 600 rpm.
SECTION: 3DRAM-seq: DpnII Digestion and biotin fill-in
31. 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 rotationspeed of 600 rpm.
SECTION: 3DRAM-seq: DpnII Digestion and biotin fill-in
32. Next day incubate samples at 62°C for 20 minutes, then cool the samples to room temperature.
SECTION: 3DRAM-seq: DpnII Digestion and biotin fill-in
33. 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 min).
SECTION: MethylHiC: Proximity ligation and decrosslinking
34. Add 720μl 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: MethylHiC: Proximity ligation and decrosslinking
35. 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
36. Add 106μl of 5M NaCl (ThermoFisher, Cat. N: AM9760G) and incubate at 68°C overnight (900 rpm 10sec, every 5 min.).
SECTION: MethylHiC: Proximity ligation and decrosslinking
37. Cool tubes at room temperature, add 1:1 volume Phenol/Chloroform/Isoamylalcohol (Invitrogen, Cat. N. 15593031), vortex for at least 30sec. and spin with maximal speed for 10minutes at 4˚C.
SECTION: MethylHiC: Proximity ligation and decrosslinking
38. Distribute the upper aqueous phase into two 2ml tubes and add to each tube 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 10-15 minutes.
SECTION: MethylHiC: Proximity ligation and decrosslinking
39. Centrifuge at maximal speed for 20 minutes at 4°C. Carefully remove the supernatant by pipetting.
SECTION: MethylHiC: Proximity ligation and decrosslinking
40. 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
41. Wash each tube with 200µl freshly prepared 70% cold ethanol and pool the samples together in one 0.5ml tube (total volume ~ 400µl, make sure the glycogen pellets are transfered). Centrifuge at maximal speed for 5 minutes at 4°C. 
SECTION: MethylHiC: Proximity ligation and decrosslinking
42. 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: 3DRAM-seq: DNA shearing (Covaris)
43. Transfer 16µl of the sample into a Covaris microTUBE-15 AFA Beads Screw-Cap tube (Covaris, Cat. N: 520145) and sheare 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: 3DRAM-seq: DNA shearing (Covaris)
44. Quantify the yield using the Qubit dsDNA High Sensitivity Assay. 
SECTION: 3DRAM-seq: Biotin removal
45. 
Mix 15µl of the sheared sample with 5µl of the Endrepair master mix.
SECTION: 3DRAM-seq: Biotin removal
46. Incubate for 4h at 20˚C followed by a heat inactivation for 20 minutes at 75˚C. Sample can be stored at -20˚C. 


SECTION: 3DRAM-seq: Bisulfite Conversion
47. NOTE: for the highest yield the libraries should be prepared directly after the bisulfite conversion. 

Prior the bisulfite conversion add roughly 0.01% GpC methylated-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: 3DRAM-seq: Bisulfite Conversion
48. Elute the bisulfite converted DNA in 31µl low EDTA TE water provided with the Accel-NGS® Methyl-Seq DNA Library Kit (Swift Bioscience, Cat. N: 30024; now xGen Methyl-Seq DNA Library Prep IDT, Cat. N.: 10009860). 
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
49. 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: 3DRAM-seq: Library construction and Biotin pulldown
50. Use 30µl of the bisulfite converted DNA for the library preparation using 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). 

Please note that one reaction volume can be used if less than 100ng of DNA was used for the bisulfite conversion.
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
51. After the ligation resuspend samples with 30 µl steril water 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: 3DRAM-seq: Library construction and Biotin pulldown
52. Wash beads 4 times with 400µl 1x Tween washing buffer for 2 minutes at 55˚C.
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
53. After the last wash remove as much as possible from the Tween washing buffer and wash beads twice with 400 µl sterile water. Thereafter resuspend beads in 50µl nuclease free water. 
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
54. Final amplification is performed with the with the EpiMark Hot Start Taq (New England Biolabs, Cat. N: M0490S) in 5 separate reactions in order to archive a higher complexity. To do so pipette 10µl of streptavidin bead solution in 5 separate PCR tubes and add 15µl of the Library amplification mix containing sample specific Methyl-Seq Set A Indexing Primers (Swift Bioscience, Cat. N: 36024; now IDT, Cat. N. 10009965 or 10005975). 
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
55. Amplify library using the following PCR program: 95˚C 30s; {95˚C 15s, 61˚C 30s, 68 ˚C 80s} x10-11; 68˚C 5min; Hold at 10˚C.
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
56. After the PCR is finished, pellet the beads and pool the library containing supernatant of different reactions of a sample together (~125µl total volume). 
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
57. Perform a 0.65x AmpureXP (Agencourt, Cat. N: A63881) purification by adding 81.25µl of room temperature AmpureXP beads to the samples followed by a incubation of 10 minutes at RT under slow rotation.  

Please note that a <0.6x AmpureXP beads can lead to the loss of the whole library ! 
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
58. Pellet the beads on a magnatic-rack, remove the supernatant and wash the beads twice with freshly prepared 80% Ethanol. 
SECTION: 3DRAM-seq: Methylation controls
2. Perform GpC methylation by mixing 20µl of the pUC19/lambda DNA mix with 30µl of the GpC methylation mix I and incubate for 1h @ 37 ˚C followed by 20 min. at 65 degree. 
SECTION: 3DRAM-seq: Methylation controls
3. Add 40ul (0.8x) of room temperature Ampure XP beads and mix well by pipetting up and down 10 times. Incubate at RT for 5min.
SECTION: 3DRAM-seq: Methylation controls
4. Separate on a magnet and remove the clear solution.
SECTION: 3DRAM-seq: Methylation controls
5. Keeping the beads on the magnet, wash twice with 200μl of 80% ethanol without mixing.
SECTION: 3DRAM-seq: Methylation controls
6. Leave the beads on the magnet for 5 minutes to allow the remaining ethanol to evaporate.
SECTION: 3DRAM-seq: Methylation controls
7. Resuspend the beads with 16µl nuclease-free water, incubate for 10 minutes at RT, pellet the beads on a magnetic-rack and transfer the DNA containing supernatant into a Covaris microTUBE-15 AFA Beads Screw-Cap tubes (Covaris, Cat. N: 520145).
SECTION: Cell fixation
19. Fixed cells are pelleted (2500xg for 5min. at 4˚C) and can be either used directly for 3DRAM-seq, ImmunoFAC-sorted or snap frozen in liquid nitrogen for storage at -80˚C. 

Please note that snap freezing leads to degradation of RNA, therefore cells for RNA isolation should be directly processed after fixation or ImmunoFACS.
SECTION: RNA-seq library construction
21. Purified RNA is quantified using Qubit RNA HS Assay Kit (ThermoFisher, Cat. N.: Q32852) and quality of the RNA is measured using the Bioanalyzer High Sensitivity RNA 6000 Pico Kit (Agilent, Cat. N.: 5067-1513).
SECTION: RNA-seq library construction
20. NOTE: this protocol was optimized for ~2x104 cells.

Pelleted cells are resuspended with 95µl nuclease free water and RNA is isolated using the Quick-RNA FFPE Miniprep (Zymo Research, Cat. N: R1008) according the manual instructions starting from the tissue digestion step.

To minimize the elution volumne Zymo-Spin IC Columns (Zymo Research, Cat. N: C1004-250) can be used.
SECTION: RNA-seq library construction
22. 100ng of high quality RNA was used for RNA library generation using the NEBNext® Single Cell/Low Input RNA Library Prep Kit (New England Biolabs, Cat. N.: E6420) according to the manual instruction.
SECTION: 3DRAM-seq: Cell lysis and GpC methylation
26. Carefully resuspend nuclei with 100µl GpC reaction buffer.
SECTION: 3DRAM-seq: Cell lysis and GpC methylation
25. Wash the nuclei pellet by adding 250µl GpC wash buffer followed by a centrifugation at 2500xg for 5 minutes (4°C) and the removal of the supernatant. 
SECTION: 3DRAM-seq: Cell lysis and GpC methylation
27. Incubate for 3 hours at 37 ˚C with slight shaking of the tubes (500 RPM) and subsidize the reaction every hour with 1µl 32mM SAM (New England Biolabs, Cat. N: M0227S) and 2µl of M.CviPI (New England Biolabs, Cat. N: M0227S).
SECTION: 3DRAM-seq: DpnII Digestion and biotin fill-in
29. Gently resuspend nuclei pellet in 40μl of 0.5% SDS (Invitrogen, Cat. N: AM9823) and incubate at 62°C for 10 minutes. Afterwards place on room temperature.
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
60. Resuspend the beads with 15µl nuclease-free water, incubate for 10 minutes at RT, 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)
SECTION: 3DRAM-seq: Library construction and Biotin pulldown
59. After the last wash remove as much as possible of the 80% Ethanol and air-dry the beads for approximately 5 minutes at RT.