Protocol SAM-Seq Zea Mays

Extraction Buffer (EB) 1

To prepare 50mlm of fresh EB1 buffer

A	B	C
2M Sucrose	10 ml	0.4M
1M Tris-HCl pH 8	0.5 ml	10mM
1M MgCl2	0.5ml	10mM
14.3M Ethidium bromide	17.5 µl	5mM
+ PIs (1 tablet per 50mls)

Extraction Buffer (EB) 2

To prepare 4ml of fresh EB2 :

A	B	C
Ethidium bromide	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 :

A	B	C
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
Ethidium bromide	1,05 µL	5mM
H20 to volume	90,3µL

Nuclear Preparation Buffer (NPB)

To prepare 10ml of fresh NPB:

A	B	C
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:

A	B	C
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:

A	B	C
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)

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 4,000 rpm 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 12,000 g for 10 minutes at 4°C. Repeat step 7 & 8 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 7 and carefully layer it on top of the clean 300µl of EB3.

Centrifuge for 45 min at 12,000 at 4 degrees.

Resuspend chromatin pellet in 300µl Nuclear Preparation Buffer (NPB) and incubate on ice for 5 min

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.

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

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.

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