TITLE: NOMe-seq of fixed cells
AUTHORS: Florian  Noack, Boyan  Bonev

[DESCRIPTION]
Protocol is based on the NOMe-seq protocol (Kelly et al.; 2012) with major modifications in the incubation times to work with fixed cells (Nordström et al.; 2019) and at the library preparation step.

[STEPS]
SECTION: Prepare control DNA
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: Prepare control DNA
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 minutes at 65 ˚C. 

SECTION: Prepare control DNA
3. Perform a 1x AmpureXP (Agencourt, Cat. N.: A63881) purification by adding 50 µl of room temperature AmpureXP beads to the samples followed by a incubation of 10 minutes at room temperature under slow rotation.  
SECTION: Prepare control DNA
7. 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: Fixation of cells
10. 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: Fixation of cells
11. 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: Fixation of cells
12. Centrifuge for 5 minutes at 500xg at 4°C. Discard supernatant into an appropriate collection container.
SECTION: Fixation of cells
13. 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 FAC-sorted, directly used for NOME-seq or pelleted (2500xg for 5 minutes at 4˚C) and then snap frozen in liquid nitrogen for storage at -80˚C.


SECTION: NOMe-seq: Cell Lysis and GpC methylation
14. NOTE: this protocol was optimized for 1x104 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: NOMe-seq: Cell Lysis and GpC methylation
15. Incubate on ice for exactly 10 minutes and centrifuge at 2500xg for 5 minutes at 4°C and remove the supernatant. 
SECTION: NOMe-seq: Cell Lysis and GpC methylation
16. Wash the nuclei pellet by adding 250µl GpC wash buffer followed by a centrifugation at 2500xg for 5 minutes at 4°C and the removal of the supernatant. 
 

SECTION: NOMe-seq: Decrosslinking and shearing
19. Add 478µl freshly prepared Proteinase-K digestion mix and incubate at 55°C for 30 minutes.
SECTION: NOMe-seq: Bisulfite Conversion and Lib. preparation
29. NOTE: for the highest yield, the libraries should be prepared directly after the bisulfite conversion and not more than 100ng of sheared DNA should be used. 

Prior the bisulfite conversion add roughly 0.05% M.CviP-treated 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: NOMe-seq: Bisulfite Conversion and Lib. preparation
30. Elute the bisulfite converted DNA in 16µl low EDTA TE water provided with the Accel-NGS® Methyl-Seq DNA Library Kit (Swift Bioscience, Cat. N.: 30024). 
SECTION: NOMe-seq: Bisulfite Conversion and Lib. preparation
32. 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 achieve a higher complexity. To do so, pipette 10µl of the purified and adapter ligated DNA 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). 
SECTION: NOMe-seq: Bisulfite Conversion and Lib. preparation
34. After the PCR is finished pool the different reactions of a sample together (~125µl total volume) and perform a 0.8x AmpureXP (Agencourt, Cat. N.: A63881) purification by adding 100µl of room temperature AmpureXP beads to the samples followed by a incubation of 10 minutes at room temperature under slow rotation.  
SECTION: NOMe-seq: Bisulfite Conversion and Lib. preparation
35. Pellet the beads on a magnetic-rack, remove the supernatant and wash the beads twice with freshly prepared 80% Ethanol. 
SECTION: Prepare control DNA
4. Pellet the beads on a magnatic-rack, remove the supernatant and wash the beads twice with freshly prepared 80% Ethanol. 
SECTION: Prepare control DNA
5. 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 control DNA
6. Resuspend the beads with 16µl 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 Covaris microTUBE-15 AFA Beads Screw-Cap tubes (Covaris, Cat. N.: 520145).
SECTION: Prepare control DNA
8. Perform another 1x AmpureXP purification as described above und elute the purified DNA in 30µl nuclease-free water. 
SECTION: Prepare control DNA
9. Quantify the yield using the Qubit dsDNA High Sensitivity Assay (ThermoFisher, Cat. N.: Q32851). Low amounts of DNA expected. Control DNA can be stored at -20˚C. 
SECTION: NOMe-seq: Cell Lysis and GpC methylation
17. Carefully resuspend nuclei with 50µl GpC reaction buffer.
SECTION: NOMe-seq: Cell Lysis and GpC methylation
18. Incubate for 3 hours at 37 ˚C with slight shaking (500 RPM) and add to the reaction every hour 0.5µl 32mM SAM (New England Biolabs, Cat. N.: M0227S) and 1µl of M.CviPI (New England Biolabs, Cat. N.: M0227S).


SECTION: NOMe-seq: Decrosslinking and shearing
20. Add 53μl of 5M NaCl (ThermoFisher, Cat. N.: AM9760G) and incubate at 68°C overnight (900rpm, 10sec, every 5 min.).
SECTION: NOMe-seq: Decrosslinking and shearing
21. 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: NOMe-seq: Decrosslinking and shearing
22. 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 10-15 minutes.
SECTION: NOMe-seq: Decrosslinking and shearing
23. Centrifuge at maximal speed for 20 minutes at 4°C. Carefully remove the supernatant by pipetting.
SECTION: NOMe-seq: Decrosslinking and shearing
24. Wash with 800μl freshly prepared 70% cold ethanol followed by centrifuge at maximal speed for 5 minutes at 4°C. 
SECTION: NOMe-seq: Decrosslinking and shearing
25. Wash with 400µl freshly prepared 70% cold ethanol and transfer everything to a 0.5ml tube (make sure glycogen pellet is transfered). Centrifuge at maximal speed for 5 minutes at 4°C. 
SECTION: NOMe-seq: Decrosslinking and shearing
26. Remove as much as possible of the ethanol and air-dry 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: NOMe-seq: Decrosslinking and shearing
27. 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: NOMe-seq: Decrosslinking and shearing
28. Quantify the yield using the Qubit dsDNA High Sensitivity Assay.
SECTION: NOMe-seq: Bisulfite Conversion and Lib. preparation
31. Use 15µl of the bisulfite converted DNA for the library preparation using the Accel-NGS® Methyl-Seq DNA Library Kit according to the manual instruction until the final amplification step. Elute the DNA after the last purification step in 50µl low EDTA TE water. 
SECTION: NOMe-seq: Bisulfite Conversion and Lib. preparation
33. Amplify library using the following PCR program: 95˚C 30s; {95˚C 15s, 61˚C 30s, 68 ˚C 60s} x10-14; 68˚C 5min; Hold at 10˚C.
SECTION: NOMe-seq: Bisulfite Conversion and Lib. preparation
36. 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: NOMe-seq: Bisulfite Conversion and Lib. preparation
37. Resuspend the beads with 15µl 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)