2.3 DNA extraction and epiGBS library preparation
We isolated genomic DNA (gDNA) from four S. cataractaepopulations (Sc1, Sc2, Sc3, Sc4) with each exposed to three treatments
(control, Cd-treated, Cu-treated) hereafter referred to as group (n = 12
groups) with each group replicated in triplicate (n = 36 samples in
total). We followed the cetyltrimethylammonium bromide (CTAB) DNA
extraction protocol for recalcitrant plant tissues
(https://www.protocols.io/view/high-quality-dna-extraction-protocol-from-recalcit-i8jchun)
with small modifications (detailed protocol available in the
Supplementary Methods - SMs). We checked the quality of the gDNA with
the NanoDrop (Nanodrop™ 8000 Spectrophotometer; Thermo Scientific), and
quantified its concentration using the Qubit 3.0 Fluorometric dsDNA BR
assay kit (Q32851; Life Technologies). We obtained high quality gDNA for
all samples except for one replicate of Sc3 in control conditions, which
we did not include in the sequencing library.
We prepared the epiGBS libraries following 71. We
digested 400 ng of gDNA from each sample with the restriction enzymePstI . Then, we ligated methylated, non-phosphorylated barcoded
adapters to both ends of the digested fragments. We concentrated the
library using the NucleoSpin™ Gel and PCR Clean-up Kit (12303368,
Macherey-Nagel™) and performed a fragment size selection with 0.8x SPRI
beads (A63880, Agencourt AMPure XP Beckman coulter). We performed nick
translation, bisulfite converted the DNA using the EZ Lightning
methylation kit (Zymo Research), and amplified the library with the KAPA
HIFI Uracil+ Hotstart Ready Mix (Roche) under the following PCR
conditions: initial denaturation step at 95ºC for 3 min; 20 cycles of
98ºC for 10s, 65ºC for 15s, and 72ºC for 15s; final extension of 72ºC
for 5 min. Finally, we quantified the library using the Qubit dsDNA
assay kit, pooled all samples using equimolar concentrations and
assessed its quality by analyzing 1 µL on a High Sensitivity DNA chip on
an Agilent 2100 Bioanalyzer. The library was sequenced at Novogene (HK)
Company Limited in Hong Kong on the Illumina HiSeq X-Ten System (PE-150
bp).