TITLE: Protocol SAM-Seq  Zea Mays
AUTHORS: basile.leduque leduque, s Quadrana Leandro

[DESCRIPTION]


Background: Epigenetic modifications, including chromatin accessibility, nucleosome positioning, and DNA methylation (5mC), are pivotal in shaping genome function. However, current short read sequencing approaches present challenges in characterising epigenetic patterns along repetitive genomic regions. 
Results: We developed Simultaneous Accessibility and DNA Methylation Sequencing (SAM-seq), a robust method utilising bacterial adenine methyltransferases (m6A-MTases) to mark accessible regions in purified plant nuclei. Coupled with Oxford Nanopore Technology sequencing, SAM-seq enables high-resolution profiling of cytosine methylation and m6A-tagged chromatin accessibility along individual chromatin fibres in A. thaliana and maize. Importantly, using naked genomic DNA we uncovered significant sequence preferences of m6A-MTases, which we show must be taken into account in order to obtain reliable accessibility profiles, particularly for the analysis of highly repetitive sequences such as centromeric repeats. Using this method we found intriguing antagonism between accessibility and DNA methylation within single molecules, somatic epigenetic variation at bivalent chromatin domains, periodicity of nucleosomal strings over centromeric repeats, and high-resolution methylation profiles over nucleosomes. 
Conclusions: Our study highlights the importance of considering intrinsic substrate preferences of DNA modifying enzymes for their use in chromatin profiling. Hence, SAM-seq is a robust and cross-species method to chart high-resolution accessibility and DNA methylation genome-wide. This method and findings will enable the investigation of chromatin-based regulation across plant species, with implications for the study of non-model plant species with limited genomic and epigenomic information. 



[STEPS]
SECTION: Plant nuclei purification  and permeabilization
9. Add the powder to 12.5 ml of Extraction Buffer (EB) 1 in a 50 ml falcon tube. Homogenise and let sit on ice for 5 min.
SECTION: Plant nuclei purification  and permeabilization
10. Add 1% Formaldehyde for crosslinking (i.e. 338 µl Formaldehyde 37% in 12.5 ml of EB1). Incubate 5 minutes on ice.
SECTION: Plant nuclei purification  and permeabilization
11. Add 1.25M glycine to stop the crosslinking (i.e. 850 ml of Glycine 2M per 338 µl Formaldehyde 37%), homogenise well before going throught the next step.
SECTION: Plant nuclei purification  and permeabilization
12. Filter the solution through Miracloth into a new 50 ml falcon tube. Repeat once.
SECTION: Plant nuclei purification  and permeabilization
13. Filter the solution through a 0.4µm filter into a new 50 ml falcon tube.
SECTION: Plant nuclei purification  and permeabilization
14. Centrifuge the filtered solution for 20 minutes at 4,000g at 4°C.
SECTION: Plant nuclei purification  and permeabilization
15. Gently remove the supernatant and resuspend the pellet in 2 ml of Extraction Buffer (EB) 2. Transfer the solution into two 1.5ml LoBind Eppendorf tubes.
SECTION: Plant nuclei purification  and permeabilization
16. Centrifuge at 11,000 g for 10 minutes at 4°C. Repeat step 15 & 16 once
SECTION: Plant nuclei purification  and permeabilization
17. Remove the supernatant and resuspend each pellet in 300 µl of Extraction Buffer (EB) 3.
SECTION: Plant nuclei purification  and permeabilization
18. In two clean 1.5 ml LoBind Eppendorf tubes, add 300 µl of EB3. Take the 300 µl solution (resuspended pellet) from step 17 and carefully layer it on top of the clean 300 µl of EB3.
SECTION: Plant nuclei purification  and permeabilization
19. Centrifuge for 45 min at 11,000g at 4 degrees.
SECTION: Plant nuclei purification  and permeabilization
20. Resuspend each chromatin pellet in 300 µl of Nuclear Preparation Buffer (NPB) and incubate on ice for 5 min.
Only one 1.5 ml LoBind Eppendorf tube of chromatin is used for the m6A-MTase reaction and is sufficient to produce enough material for one promethION run; the second tube can be stored at -80°C.
SECTION: m6A-MTases reaction
21. Spin down nuclei at 1000g for 5 min at 4°C and resuspend in 300 µl of Plant-Tween-Wash-Buffer (PTWB).
SECTION: m6A-MTases reaction
22. Spin down nuclei at 1000g for 5 min at 4°C and resuspend pellet in 380 µl 1X rCutSmart, add 10 µl SAM (32mM) and 6 µl EcoGII (i.e. 7.5U for 100 µl). Incubate 30 minutes at 30°C 
SECTION: m6A-MTases reaction
23. Add  20 µl NaCl (5M) and incubate overnight at 65 °C (or 1h30 if the chromatin has not been cross-linked).
SECTION: m6A-MTases reaction
24. Add 5 µl of RNAse A (20mg/ml) and 2 µl Proteinase K (20mg/ml). Incubate 30 minutes at 42°C.
SECTION: Phenol/Chloroform extraction of genomic DNA
25. Add 1 volume of 1:1 phenol:chloroform (i.e. 400 µl) 
SECTION: Phenol/Chloroform extraction of genomic DNA
27. Recover the aqueous phase (upper) in a new 1.5 ml LoBind Eppendorf tube
SECTION: Phenol/Chloroform extraction of genomic DNA
28. Add 1 volume of chloroform (i.e. 400 µl)
SECTION: Phenol/Chloroform extraction of genomic DNA
29. Centrifuge at 12,000 g for 10 minutes at 4°C.
SECTION: Phenol/Chloroform extraction of genomic DNA
30. Recover aqueous phase (upper) in a new 1.5 ml LoBind Eppendorf tube
SECTION: Phenol/Chloroform extraction of genomic DNA
31. Precipitate DNA by adding 1/10 volume of NaAcétate 3M pH5,2 (ie 40 µl) and 2,5 volume of EtOH 100% (ie 1 ml)
SECTION: Phenol/Chloroform extraction of genomic DNA
32. Incubate at –20°C from 45 minutes to overnight.
SECTION: Phenol/Chloroform extraction of genomic DNA
33. Centrifuge at 12000g for 30 minutes at 4°C to precipitate DNA.
SECTION: Phenol/Chloroform extraction of genomic DNA
34. Wash the pellet with 300 µl EtOH 70%.
SECTION: Phenol/Chloroform extraction of genomic DNA
35. Remove EtOH and dry pellets.
SECTION: Phenol/Chloroform extraction of genomic DNA
36. Elute in 20 µl Tris-HCl 10mM pH 8.0
SECTION: Phenol/Chloroform extraction of genomic DNA
26. Centrifuge at 12,000 g for 10 minutes at 4°C
SECTION: Library preparation
37. 
Follow the protocol provided by Oxford Nanopore technology ( Genomic_DNA_by_Ligation_SQK-LSK110) with the following modification:

-Use 0.3 to 1 µg of DNA per library 
-For a better library preparation increase the End-Prep step up to 20 minutes at 20°C and Ligation step incubation to at least 1h
- Elute in Tris-HCl 10mM pH 8.0.
- LFB was used for final size selection step
- Quantify DNA using the Qubit dsDNA HS Assay Kit (Q33230) to check yield.








SECTION: Sequencing
38. Sequence during 72h on v9.4.1 flow cell  FLO-MIN106.1 or FLO-PRO002 in MinION or PromethION sequencer, respectively.



SECTION: Reagent Preparation
1. Extraction Buffer (EB) 1 
To prepare 50 ml of fresh EB1 buffer

   Extraction Buffer (EB) 1for 50 ml:Final Concentration2M sucrose10 ml 0.4M 1M Tris-HCl pH 80.5 ml10mM 1M  MgCl20.5ml 10mM14.3M  BME17.5 µl 5mM+ PIs (1 tablet  per 50mls)
Fill to 50 ml with distilled water 
SECTION: Reagent Preparation
2. Extraction Buffer (EB) 2
To prepare 4 ml of fresh EB2 :
 


SECTION: Reagent Preparation
3. Extraction Buffer (EB) 3
 To prepare 3 ml of fresh EB 3 :
  
   Extraction Buffer (EB) 33 mLFinal Concentration2M sucrose2550 µL sucrose 1.7M 1M  Tris-HCl pH 830 µL Tris HCl pH810mM  20% Triton X-10025 µL Triton0.15%  1M MgCl26 µL MgCl22mM BME1,05 µL BME 5mM + Pis (1 mini-tablet diluted in 1 ml)300 µL Mini complete Pi'sH20 to volume90,3µL 

SECTION: Reagent Preparation
4. 


Nuclear Preparation Buffer (NPB)
  To prepare 10 ml of fresh NPB: 

   Nuclear Preparation Buffer (NPB)Volume for 10mlFinal Concentration0.5M MOPS   pH7400µl20 mM 5M NaCl80 µl40 mM 1M KCl900µl90 mM 0.5 M EDTA40µl2 mM 250mM EGTA20 µl0.5 mM 3.5M spermidine1.2µl0.5 mM 70 mM spermine28.5µl0.2 mM 
Fill to 10 ml with distilled water 
SECTION: Reagent Preparation
5. Plant-Tween-Wash-Buffer (PTWB)
To prepare 3ml of fresh PTWB:
 
   20% Tween-2030 µl 0.2% Tween-201M pH7,5 HEPES-KOH60 µl 20 mM pH7,5 HEPES-KOH5M NaCl90 µl 150 mM NaClPI Mini300 µlPI 1/10Stock solution 3.5M0,4 µlSpermidine 0,5 mM 2530 µlH20

SECTION: Reagent Preparation
6. 

 Activation Buffer (AB)
To prepare 3ml of freash AB:
 
   1M Tris-HCl pH 8750 µl15 mM5M NaCl150 µl15 mM1M KCl3 ml60 mM0.5 M EDTA100µl1 mM250mM EGTA100 µl0.5 mM 3.5M spermidine0.715 µl0.05 mMBSA50 mg0.1%H2Ofill to 50 mlSAM 32 mM(add before reaction) 800µM
Add enough distilled water to raise the volume to 3 mL.
SECTION: Reagent Preparation
7. Reagents:
1.5 ml Eppendorf DNA LoBind tubes (022431021 Fisher)
Pierce, protease inhibitor without EDTA (A32965 Thermofisher)
Pierce, mini-tablets protease inhibitor without EDTA (A32955 Thermofisher)
for m6A reaction
EcoGII/ rCutSmart/ SAM (M0603S New England Biolabs)
Proteinase K  (25530049 Thermofisher)
RNAse A  (T3018L New England Biolabs)
for library preparation:
NEBNext Companion Module for Oxford Nanopore Ligation Seq (E7180S New England Biolabs)
SQK-LSK110 (Oxford Nanopore Technologies)
for nanopore sequencing:
FLO-MIN106.1 or  FLO-PRO002  (Oxford Nanopore Technologies)



SECTION: Plant nuclei purification  and permeabilization
8. Starting material: 0.75 g of grinded powder (7 days old leaves)