Zymo ez dna methylation gold kit

Extracted DNA was bisulphite-converted with ZYMO EZ DNA Methylation-Gold Kit according to the manufacturer's protocol (Zymo Research, Orange, CA, USA). The bisulphite-modified DNA was resuspended in 10 μl of TE buffer for the following methylation analysis.

Gene-specific DNA methylation was assessed by a next generation sequencing-based BSP, according to previously published method (Gao et al., 2014 (link)). In brief, BSP primers were designed using the online MethPrimer software and listed in Supplementary Table 1. A total of 1 μg of genomic DNA was converted using the ZYMO EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) and one twentieth of the elution products were used as templates for PCR amplification with 35 cycles using KAPA 2G Robust HotStart PCR Kit (Kapa Biosystems, Wilmington, MA, USA). For each sample, BSP products of multiple genes were pooled equally, 5′-phosphorylated, 3′-dA-tailed and ligated to barcoded adapter using T4 DNA ligase (NEB). Barcoded libraries from all samples were sequenced on Illumina platform. For the bisulfite sequencing reads of each sample, firstly, adapters and low-quality reads were removed using software Trimmomatic-0.36. After removing the adapter sequences and filtering out the low-quality reads, the clean sequencing reads were directly aligned to the target sequences using software Bsmap (v2.73) with the default parameters which combines genome hashing and bitwise masking to achieve fast and accurate bisulfite mapping. Methylation level of C base in TYLCV genome was calculated as follows.

Each sample is a mixture of three or more plant tissues and the DNA was isolated using a cetyltrimethylammonium bromide method (CTAB) plant genomic kit (Aidlab, Peking, China). The gDNA was checked for integrity by agarose gel electrophoresis and the concentration was measured using a non-ultraviolet method. The DNA was fragmented using a Diagenode sonicator to a mean size of 100–300 bp, followed by DNA-end repair, 3’-dA overhangs and the ligation of methylated sequencing adaptors according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). DNA samples were bisulfite-treated using the ZYMO EZ DNA Methylation-Gold Kit (ZYMO Research, Irvine, CA, USA). The bisulfite-converted DNA was then amplified by PCR and purified after desalting and size selection. Quantified and converted DNA libraries were loaded onto the Illumina HiSeq2000 platform and paired-end sequencing was performed to generate paired-end 125-bp reads for each library. The sequencing was carried out without biological replicate.

Genomic DNA was extracted from muscle from HSS participants using the QIAamp DNA mini kit (Qiagen) and from HSSe participants using the high salt method.²⁴ A total of 750 ng of genomic DNA was treated with sodium bisulfite using Zymo EZ DNA Methylation‐Gold kit (ZymoResearch, USA) and hybridized to the Infinium Human MethylationEPIC BeadChip array (Illumina, Inc., USA) at the Centre for Molecular Medicine and Therapeutics (http://www.cmmt.ubc.ca).