Epignome methyl seq kit

Genomic DNA was isolated from two to three cauline leaves formed following glyphosate exposure from individual plants in quadruplicate for each of the three treatment levels (0%, 5%, and 10%) using the Biosprint-15 plant DNA extraction kit (Qiagen, Hilden, Germany). The 12 samples were sent to Genomics Research Laboratory at Biocomplexity Institute of Virginia Tech (Blacksburg, VA, USA) for library preparation and bisulfite sequencing. 100 ng of intact DNA was bisulfite converted using EZ DNA Methylation-Gold Kit (#D5005; Zymo Research, Irvine, CA, USA), following the manufacturers protocol, except eluting into 9 ul. The entire amount of the purified bisulfite-treated DNA was converted to Illumina DNA libraries using EpiGnome Methyl-Seq kit (Epicentre, Madison, WI, USA). Six samples each were individually barcoded, quantitated by qPCR, and pooled to sequence on the entire Illumina HiSeq Rapid Run flowcell. Libraries were clustered on-board at a concentration of 8.5 pM with 3% phiX, onto a flow cell using Illumina’s HiSeq Rapid Paired End Cluster Kit V2 (PE-402-4002), and sequenced 2× 101 cycles using HiSeq Rapid SBS Kit (200-cycles) (FC-402-4021).

WGBS libraries were prepared using the EpiGnome Methyl‐Seq Kit (Epicentre, EGMK81312), according to the manufacturer's protocol. Library quality was assessed with the Agilent 2100 Bioanalyzer using the High‐Sensitivity DNA Kit (Agilent, CA, USA). DNA was quantified using the KAPA Library Quantification Kit by quantitative PCR (KAPA 6 Biosystems). Libraries from patient specimens were analysed with 70 bp paired‐end sequencing on the Illumina HiSeq 2500 platform using TruSeq Rapid SBS Kit ‐ HS (50 cycle) and TruSeq Rapid PE Cluster Kit ‐ HS. Six samples were multiplexed across two lanes. Sequencing was performed multiple times to gain sufficient coverage.

DNA from rhesus, cow, horse, dog, squirrel monkey, and mouse placenta tissue was purified using Qiagen's Puregene kit. MethylC-seq libraries were made as described previously [21 (link)]. Briefly, the genomic DNA was sonicated to ~300 bp and methylated Illumina adapters were ligated to the ends. The library was bisulfite converted, amplified for 14 cycles, and sequenced on either an Illumina HiSeq or GAII. For the opossum EEM, rhesus trophoblast, human cord blood, and cow, dog, and opossum brain samples, MethylC-seq libraries were made using the Epicentre EpiGnome Methyl-Seq kit according to the manufacturer's recommendations except that 14 cycles of amplification were performed. For cow oocytes, DNA from 100 cells was purified using Zymo's Quick-gDNA MicroPrep kit and the MethylC-seq libraries were prepared using Zymo's Pico Methyl-seq Library Prep Kit according to the manufacturer's instructions. After sequencing on HiSeq2500/2000 machines, reads were mapped to the respective genomes using BS Seeker [50 (link)] and only one read per genomic position was kept to prevent clonal PCR amplification biases. CpG site methylation data were combined from both DNA strands. Because methylation data was analyzed over large genomic distances and/or smoothed, no minimum coverage was required for CpG sites used in this analysis [35 (link)].

Genomic DNA was extracted from three dnmt1^+/+ and three dnmt1^−/− zebrafish of G2 generation at 18 dpf, sperm of three dnmt1^+/+ and three dnmt1^−/− zebrafish of G2 generation, and sperm of three G4* and three G4+ fish of G4 generation using the DNeasy blood and tissue kit (Qiagen). 1 μg and 0.5 μg of DNA was used for bisulfite reactions and library construction using the TruSeq DNA PCR-free library preparation kit (Illumina) and the EpiGnome Methyl-Seq kit (Epicentre), respectively. The fragments were sequenced in paired-end 100 bp mode on 1 lane of Illumina HiSeq 2500 instrument.

Sequencing libraries for DNA methylation mapping were prepared using the µWGBS protocol^{24 (link)}. Starting directly from lysed cells in digestion buffer, proteinase K digestion was performed at 50 °C for 20 minutes. Custom-designed methylated and unmethylated oligonucleotides were added at a concentration of 0.1% to serve as spike-in controls for monitoring bisulfite conversion efficiency. Bisulfite conversion was performed using the EZ DNA Methylation-Direct Kit (Zymo Research, D5020) according to the manufacturer’s protocol, with the modification of eluting the DNA in only 9 µl of elution buffer. Bisulfite-converted DNA was used for single-stranded library preparation using the EpiGnome Methyl-Seq kit (Epicentre, EGMK81312) with the described modifications. Quality control of the final library was performed by measuring DNA concentrations using the Qubit dsDNA HS assay (Life Technologies, Q32851) on Qubit 2.0 Fluorometer (Life Technologies, Q32866) and by determining library fragment sizes with the Agilent High Sensitivity DNA Analysis kit (Agilent, 5067-4626) on Agilent 2100 Bioanalyzer Station (Agilent, G2939AA). All libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the 2x75bp paired-end setup on the Illumina HiSeq 3000/4000 platform.