Protocol Citation: basile.leduque leduque, s Quadrana Leandro 2024. Protocol SAM-Seq Zea Mays. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3x25zg25/v2Version created by basile.leduque leduque
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 28, 2023
Last Modified: May 02, 2024
Protocol Integer ID: 99097
Funders Acknowledgement:
European Research Council
Grant ID: 948674
Abstract
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.
Reagent Preparation
Reagent Preparation
Extraction Buffer (EB) 1
To prepare 50mlm of fresh EB1 buffer
| Component | Amount | Final Concentration | |
| 2M Sucrose | 10 ml | 0.4M | |
| 1M Tris-HCl pH 8 | 0.5 ml | 10mM | |
| 1M MgCl2 | 0.5ml | 10mM | |
| 14.3M 2-mercaptoethanol | 17.5 µl | 5mM | |
| + PIs (1 tablet per 50mls) |
Extraction Buffer (EB) 2
To prepare 4ml of fresh EB2 :
| Component | Amount | Final Concentration | |
| 14.3M 2-mercaptoethanol | 1,4 µL | 5mM | |
| 20% Triton X-100 | 200 µL | 1% | |
| H20 | 2818,6 µL | ||
| 1M MgCl2 | 40 µL | 10mM | |
| 1M Tris-HCl pH 8 | 40 µL | 10mM | |
| PIs (1 mini-tablet diluted in 1 ml) | 400 µL | ||
| 2M Sucrose | 500 µl | 0.25M |
Extraction Buffer (EB) 3
To prepare 3ml of fresh EB 3 :
| Component | Amount | Final Concentration | |
| 1M Tris-HCl pH 8 | 30 µL | 10mM | |
| 1M MgCl2 | 6 µL | 2mM | |
| 20% Triton X-100 | 25 µL | 0.15% | |
| + Pis (1 mini-tablet diluted in 1 ml) | 300 µL | ||
| 2M sucrose | 2550 µL | 1.7M | |
| 1,05 µL | 5mM | ||
| H20 to volume | 90,3µL |
Nuclear Preparation Buffer (NPB)
To prepare 10ml of fresh NPB:
| Component | Amount | Final Concentration | |
| 0.5M MOPS pH7 | 400µl | 20 mM | |
| 5M NaCl | 80 µl | 40 mM | |
| 1M KCl | 900µl | 90 mM | |
| 0.5 M EDTA | 40µl | 2 mM | |
| 250mM EGTA | 20 µl | 0.5 mM | |
| 3.5M spermidine | 1.2µl | 0.5 mM | |
| 70 mM spermine | 28.5µl | 0.2 mM | |
| 1 mini-tablet diluted in 1ml | 1ml | 1X |
Plant-Tween-Wash-Buffer (PTWB)
To prepare 3ml of fresh PTWB:
| Component | Amount | Final Concentration | |
| 20% Tween-20 | 30 µl | 0.2% | |
| 1M pH7,5 HEPES-KOH | 60 µl | 20 mM | |
| 5M NaCl | 90 µl | 150 mM | |
| PI Mini | 300 µl | ||
| Spermidine 3.5M | 0,4 µl | 0,5 mM | |
| H2O | 2530 µl |
Activation Buffer (AB)
To prepare 3ml of freash AB:
| Component | Amount | Final Concentration | |
| 1M Tris-HCl pH 8 | 750 µl | 15 mM | |
| 5M NaCl | 150 µl | 15 mM | |
| 1M KCl | 3 ml | 60 mM | |
| 0.5 M EDTA | 100µl | 1 mM | |
| 250mM EGTA | 100 µl | 0.5 mM | |
| 3.5M spermidine | 0.715 µl | 0.05 mM | |
| BSA | 50 mg | 0.1% | |
| H2O | fill to 50 ml | ||
| SAM 32 mM | (add before reaction) | 800µM |
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)
Plant nuclei purification and permeabilization
Plant nuclei purification and permeabilization
Starting material: 0.75 g of powder (7 days old leaves)
Add the powder to 12.5 ml of Extraction Buffer (EB) 1 in a 50 ml falcon tube. Let sit on ice for 5 min.
Add 1% Formaldehyde for crosslinking (i.e. 338 µl Formaldehyde 37% in 12.5ml of EB1). Incubate 5 minutes
Add 1.25 M glycine to stop the crosslinking (i.e. 850ml of Glycine 2M per 338 µl Formaldehyde 37%)
Filter the solution through Miracloth into a new 50 ml falcon tube. Repeat once.
Filter the solution through a 0.4µm filter into a new 50 ml falcon tube.
Centrifuge the filtered solution for 20 minutes at 4,000g at 4°C.
Gently remove supernatant and resuspend the pellet in 2 ml of Extraction Buffer (EB) 2. Transfer the solution into one 1.5ml Eppendorf tube.
Centrifuge at 11,000 g for 10 minutes at 4°C. Repeat step 15 & 16 once
Remove supernatant and resuspend pellet in 300µl of Extraction Buffer (EB) 3.
In a clean Eppendorf tube, add 300ul of EB3. Take the 300µl solution (resuspended pellet) from step 17 and carefully layer it on top of the clean 300µl of EB3.
Centrifuge for 45 min at 11,000g at 4 degrees.
Resuspend chromatin pellet in 300µl Nuclear Preparation Buffer (NPB) and incubate on ice for 5 min
m6A-MTases reaction
m6A-MTases reaction
Spin down nuclei at 1000g for 5 min at 4°C and resuspend in Plant-Tween-Wash-Buffer (PTWB).
Resuspend pellet in 38 µl 1X rCutSmart, containing 10µl SAM and 6µl EcoGII (i.e. 7.5U for 100µl). Incubate 30 minutes at 30°C
Add 20 µl NaCl (5M) and incubate overnight at 65 °C (or 1h30min if not crosslinked).
Add 5µl of RNAse A (20mg/ml) and 2µl Proteinase K. Incubate 30 minutes at 42°C.
Phenol/Chloroform extraction of genomic DNA
Phenol/Chloroform extraction of genomic DNA
Add 1 volume of 1:1 phenol:chloroform (i.e. 400µl)
Centrifuge at 12,000 g for 10 minutes at 4°C
Recover the aqueous phase (upper) in a new LoBind Eppendorf tube
Add 1 volume of chloroform (i.e. 400µl)
Centrifuge at 12,000 g for 10 minutes at 4°C.
Recover aqueous phase (upper) in a new LoBind Eppendorf tube
Precipitate DNA by adding 1/10 volume of NaAcétate 3M pH5,2 + 2,5 volume of EtOH 100%
Incubate at –20°C from 45 minutes to overnight.
Centrifuge at 12000g for 30 minutes at 4°C to precipitate DNA.
Wash the pellet with 300µ EtOH 70%.
Remove EtOH and dry pellets.
Elute in 20µl TE pH 8.0
Library preparation
Library preparation
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.
Sequencing
Sequencing
Sequence during 72h on v9.4.1 flow cell FLO-MIN106.1 or FLO-PRO002 in MinION or PromethION sequencer, respectively.