Sample preparation for genome wide DNA methylation analysis 

a) Grow seeds of soybean genotype Williams 82 in a Miracle-Gro Moisture Control potting medium at 25°°C, 16 h d−1 light at 230 to 365 μM m−2 s−1, and 90% relative humidity in a growth chamber.

b) Collect a trifoliate leaf at the V2 developmental stage for DNA isolation.

c) Isolate genomic DNA (gDNA) using the Zymo Research Quick-DNA Plant/Seed Miniprep kit (Cat #D6020; Irvine, CA, USA).

d) Mesure DNA Concentration using a Qubit fluorometer (Thermo Fisher Scientific; Waltham, MA, USA) coupled with a ThermoFisher Scientific dsDNA High Sensitivity Assay kit (Cat #Q32851 ).

e) Assess DNA purity from 260/230 and 260/280 nm absorbance ratios using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).

f) Determine the quality and size of obtained DNA using gel electrophoresis (1% agarose gel with 1X Tris acetate-EDTA buffer).

g) Prepare a non-methylated control by spiking the soybean gDNA with Escherichia coli gDNA (Zymo Research, Cat #D5016) representing 1% of the total gDNA input.

h) Separate the spiked sample into three aliquots (each 65 μL), and shear the samples using Qsonica sonicators (Newtown, CT, USA).

i) Use sonication settings as 15 sec on/90 sec off for 8 cycles at 20 kHz frequency, resulting in DNA fragments of approximately 350 bp.

j) Follow library preparation as described below with the sheared aliquots.

a) Prepare sequencing libraries using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs; Cat #E7645S; Ipswich, MA, USA) following manufacturer’s instruction.

b) Use a total of 200 ng (for NEB EM-seq kit) or 1 μg (Zymo and QIAGEN Conversion kits) of starting DNA based on the manufacturer’s instructions.

c) Use NEBNext® Multiplex Oligos for Illumina (NEB Cat #E7535S/L) to repair the ends of sheared DNA and then to ligate Illumina Methylated Adaptors.

d) Now, purify the adapter-ligated fragments using Solid Phase Reversible Immobilization (SPRI) magnetic beads (Beckman Coulter Inc.; Cat #B23317; Brea, CA, USA).

e) For the cytosine conversion of adapter-ligated purified DNA samples, prepare samples following manufacturer’s instructions using Zymo Conversion kit (EZ DNA Methylation™ Kit, Zymo Research; Cat #D5001), QIAGEN Conversion kit (EpiTect Bisulfite Kit, QIAGEN; Cat #59104), or NEB EM-seq kit (NEBNext® Enzymatic Methyl-seq Conversion Module, New England Biolab; Cat #E7125S/L).

f) For the Zymo Conversion kit (EZ DNA Methylation™ Kit, Zymo Research; Cat #D5001), incubate the DNA sample with CT Conversion Reagent (Zymo Research; Cat #D5001-1) in a thermocycler for approximately 16 hrs using following PCR settings.

A	B	C	D	E
EZ DNA Methylation Kit (Zymo Research)
Step	Time	Temperature	Cycles	Total time required for cytosine conversion
Denaturation	30 sec	95°C	55	≈ 16 hr.
Bisulfite conversion	15 min	50°C
Hold	∞	4°C

g) Treat the resulting sample with M-Desulphonation Buffer from the kit for desulphonation (Zymo Research; Cat #D5001-5).

h) For the QIAGEN Conversion kit (EpiTect Bisulfite Kit, QIAGEN; Cat #59104), incubate the sample with bisulfite mix and the DNA protection buffer provided in the kit in the thermocycler for approximately 5 hrs using the following PCR settings.

A	B	C	D	E
Epitech Bisulfite Conversion kit (Qiagen)
Step	Time	Temperature	Cycles	Total time required for cytosine conversion
Denaturation	5 min	95°C	1	≈ 5 hr.
Incubation	25 min	60°C
Denaturation	5 min	95°C
Incubation	1hr 25 min	60°C
Denaturation	5 min	95°C
Incubation	2h 55 min	60°C
Hold	∞	20°C

i) Treat the converted DNA samples with Buffer BD from the kit for desulphonation.

j) For the NEB EM-seq kit (NEBNext® Enzymatic Methyl-seq Conversion Module, New England Biolab; Cat #E7125S/L), follow the manufacturer’s instructions using TET2 (NEB Cat #E7130AVIAL) and APOBEC (NEB Cat #E7133AVIAL) enzymes in a series of steps, which ultimately converts cytosines to uracil, allowing for the detection of 5 mC and 5 hMC in the DNA sample. Use the following PCR settings.

A	B	C	D	E
NEBNext Enzymatic Methyl Seq Conversion Module (NEB)
Step	Time	Temperature	Cycles	Total time required for cytosine conversion
Oxidation of 5-methylcytosines and 5- Hydroxymethylcytosines	1hr 30 min	37°C	1	≈ 5 hr.
Denaturation of DNA	10 min	50°C
Deamination of Cytosines	3hr.	37°C
Hold	∞	4°C

k) Purify the cytosine converted DNA samples using Solid Phase Reversible Immobilization (SPRI) magnetic beads (Beckman Coulter Inc.; Cat #B23317; Brea, CA, USA).

l) Now, use the purified-cytosine converted samples to prepare illumine sequencing samples with NEBNext® Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1) (NEB Cat #E7535S/L) using EpiMark® Hot Start Taq DNA Polymerase (NEB Cat # M0490S) following manufacturer instructions with following PCR settings.

A	B	C	D
Step	Time	Temperature	Cycles
Initial denaturation	30 sec	95°C	16
Extension	15 sec 30 sec 30 sec	90°C  61°C  68°C
Hold	∞	4°C

a) Purify the illumine sequencing libraries using Solid Phase Reversible Immobilization (SPRI) magnetic beads (Beckman Coulter Inc.; Cat #B23317; Brea, CA, USA).

b) Measure the concentration and quality of the libraries using Thermo-Invitrogen Qubit Fluorometer, NanoDrop ND-1000 spectrophotometer, Agilent Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA).

c) Sequence the libraries using an Illumina Hi-Seq sequencer to obtain 150 bp paired-end reads.

Analysis code available from: https://github.com/ajwije/DNA_methylation_analysis.git