TITLE: Protocol SAM-Seq  A.Thaliana
AUTHORS: basile.leduque, 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
7. Starting material: 1g of powder ( Aerial part of ~17 days seedling )

Add the powder to 25 ml of Extraction Buffer (EB) 1 in a 50 ml falcon tube. Let sit on ice for 5 min.
SECTION: Plant nuclei purification  and permeabilization
8. Add 1% Formaldehyde for crosslinking (i.e. 675 µl Formaldehyde 37% in 25ml of EB1). Incubate 5 minutes
SECTION: Plant nuclei purification  and permeabilization
9. Add 1.25 M glycine to stop the crosslinking (i.e. 1.7ml of Glycine 2M per 675 µl Formaldehyde 37%)
SECTION: Plant nuclei purification  and permeabilization
10. Filter the solution through Miracloth into a new 50 ml falcon tube. Repeat once.
SECTION: Plant nuclei purification  and permeabilization
11. Filter the solution through a 0.4µm filter into a new 50 ml falcon tube.
SECTION: Plant nuclei purification  and permeabilization
12. Centrifuge the filtered solution for 20 minutes at 4,000 rpm at 4°C.
SECTION: Plant nuclei purification  and permeabilization
13. Gently remove supernatant and resuspend the pellet in 2 ml of Extraction Buffer (EB) 2. Transfer the solution into four 1.5ml Eppendorf tubes.
SECTION: Plant nuclei purification  and permeabilization
14. Centrifuge at 12,000 g for 10 minutes at 4°C. Repeat step 7 & 8 once
SECTION: Plant nuclei purification  and permeabilization
15. Remove supernatant and resuspend each pellet in 300µl of Extraction Buffer (EB) 3.
SECTION: Plant nuclei purification  and permeabilization
16. In a clean Eppendorf tube, add 300ul of EB3. Take the 300µl solution (resuspended pellet) from step 7 and carefully layer it on top of the clean 300µl of EB3.
SECTION: Plant nuclei purification  and permeabilization
17. Centrifuge for 45 min at 12,000 at 4 degrees.
SECTION: Plant nuclei purification  and permeabilization
18. Resuspend each chromatin pellet in 300µl Nuclear Preparation Buffer (NPB) and incubate on ice for 5 min
SECTION: m6A-MTases reaction
19. Spin down nuclei at 1000g for 5 min at 4°C and resuspend in Plant-Tween-Wash-Buffer (PTWB).
SECTION: m6A-MTases reaction
20. Resuspend each pellet in 193.5 µl 1X rCutSmart, containing 5µl SAM and 1.5µl EcoGII (i.e. 3.75U for 100µl). Incubate 30 minutes at 30°C
SECTION: m6A-MTases reaction
21. Add 200 µl rSmartCut 1X + 20 µl NaCl (5M) and incubate overnight at 65 °C (or 1h30min  if not crosslinked).
SECTION: m6A-MTases reaction
22. Add 5µl of RNAse A (20mg/ml) and 2µl Proteinase K. Incubate 30 minutes at 42°C.
SECTION: Phenol/Chloroform extraction of genomic DNA
23. Add 1 volume of 1:1 phenol:chloroform (i.e. 400µl) 
SECTION: Phenol/Chloroform extraction of genomic DNA
25. Recover the aqueous phase (upper) in a new LoBind Eppendorf tube
SECTION: Phenol/Chloroform extraction of genomic DNA
26. Add 1 volume of chloroform (i.e. 400µl)
SECTION: Phenol/Chloroform extraction of genomic DNA
27. Centrifuge at 12,000 g for 10 minutes at 4°C.
SECTION: Phenol/Chloroform extraction of genomic DNA
28. Recover aqueous phase (upper) in a new LoBind Eppendorf tube
SECTION: Phenol/Chloroform extraction of genomic DNA
29. Precipitate DNA by adding 1/10 volume of NaAcétate 3M pH5,2 + 2,5 volume of EtOH 100%
SECTION: Phenol/Chloroform extraction of genomic DNA
30. Incubate at –20°C from 45 minutes to overnight.
SECTION: Phenol/Chloroform extraction of genomic DNA
31. Centrifuge at 12000g for 30 minutes at 4°C to precipitate DNA.
SECTION: Phenol/Chloroform extraction of genomic DNA
32. Wash the pellet with 300µ EtOH 70%.
SECTION: Phenol/Chloroform extraction of genomic DNA
33. Remove EtOH and dry pellets.
SECTION: Phenol/Chloroform extraction of genomic DNA
34. Elute in 20µl TE pH 8.0
SECTION: Phenol/Chloroform extraction of genomic DNA
24. Centrifuge at 12,000 g for 10 minutes at 4°C
SECTION: Library preparation
35. 
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 10 mM.
- LFB was used for final size selection step
- Quantify DNA using the Qubit dsDNA HS Assay Kit (Q33230) to check yield.








SECTION: Sequencing
36. 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 50mlm 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)

SECTION: Reagent Preparation
2. Extraction Buffer (EB) 2
To prepare 4ml of fresh EB2 :
 
   2Msucrose500 µL sucrose 0.25M 1M Tris-HCl pH 840 µL Tris HCl pH8 10mM 1M MgCl240 µL MgCl2 10mM 20% Triton X-100200 µL Triton 1% 14.3M BME1,4 µL BME5mM PIs (1 mini-tablet diluted in 1 ml)400 µL Mini complete Pi'sH202818,6 µL H2O

SECTION: Reagent Preparation
3. Extraction Buffer (EB) 3
 To prepare 3ml 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 10ml 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 

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. Reagents:

for m6A reaction
EcoGII/ rCutSmart/ SAM (M0603S 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)