Truseq nano library synthesis kits

DNA libraries were prepared from methyl-sensitive restriction endonuclease (MSRE) [30 (link), 31 (link)] fragmented genomic DNA (gDNA) using HpaII, which recognizes C (CpG) G sites. A standard sequencing protocol was then performed including randomized shearing (Covaris, Woburn, MA) and synthesis of a gDNA fragment library using Illumina TruSeq Nano library synthesis kits (San Diego, CA). Next generation sequencing (NGS) was performed on an Illumina ×10 platform by Macrogen USA (Rockville, MD). The protocol generated single end reads (150 bp) with >20× coverage of the regions captured. FASTQ data files were processed to calculate the probability of methylation at individual CpG sites through a commercial bioinformatics pipeline and software platform (Genome Profiling, Newark, DE). For convenience, the term “CpG” in this paper refers to “C (CpG) G” HpaII restriction sites. Validation of the HpaII approach was carried out using “spike in” DNA sequences synthesized with known methyl-CpG composition (see Additional file 1: Figure S1).

Genomic DNA was isolated using Gentra Puregene kits (Qiagen, Germantown, MD, USA). A previously published DNA methylation assay [39 (link),55 (link),56 (link)] was utilized. Briefly, DNA libraries for next generation sequencing (NGS) were prepared by digesting genomic DNA with methyl–sensitive restriction endonuclease HpaII, which recognizes CCGG sites. A standard sequencing protocol was then performed including randomized shearing (Covaris, Woburn, MA, USA) and synthesis of a gDNA fragment library using Illumina TruSeq Nano library synthesis kits (San Diego, CA, USA). NGS was performed on an Illumina ×10 platform by Psomagen (Rockville, MD, USA). The protocol generated single end reads (150 bp) with >20× coverage of the regions captured. FASTQ data files were processed to calculate the probability of methylation at individual CpG sites through a commercial bioinformatics pipeline and software platform (Genome Profiling LLC, Newark, DE, USA). For convenience, the term “CpG” in this paper refers to “C(CpG)G” HpaII restriction sites.