RRC ID 78299
Author Tanaka Y, Mizuguchi R, Koseki N, Suzuki H, Suzuki T.
Title Quality assessment of enzymatic methyl-seq library constructed using crude cell lysate.
Journal Biochem Biophys Res Commun
Abstract Enzymatic methyl-seq (EM-seq), an enzyme-based method, identifies genome-wide DNA methylation, which enables us to obtain reliable methylome data from purified genomic DNA by avoiding bisulfite-induced DNA damage. However, the loss of DNA during purification hinders the methylome analysis of limited samples. The crude DNA extraction method is the quickest and minimal sample loss approach for obtaining useable DNA without requiring additional dissolution and purification. However, it remains unclear whether crude DNA can be used directly for EM-seq library construction. In this study, we aimed to assess the quality of EM-seq libraries prepared directly using crude DNA. The crude DNA-derived libraries provided appropriate fragment sizes and concentrations for sequencing similar to those of the purified DNA-derived libraries. However, the sequencing results of crude samples exhibited lower reference sequence mapping efficiencies than those of the purified samples. Additionally, the lower-input crude DNA-derived sample exhibited a marginally lower cytosine-to-thymine conversion efficiency and hypermethylated pattern around gene regulatory elements than the higher-input crude DNA- or purified DNA-derived samples. In contrast, the methylation profiles of the crude and purified samples exhibited a significant correlation. Our findings indicate that crude DNA can be used as a raw material for EM-seq library construction.
Volume 696
Pages 149488
Published 2024-2-12
DOI 10.1016/j.bbrc.2024.149488
PII S0006-291X(24)00023-8
PMID 38219485
MeSH Base Sequence Cloning, Molecular DNA* / analysis DNA* / genetics DNA Methylation* Gene Library High-Throughput Nucleotide Sequencing / methods Sequence Analysis, DNA / methods Sulfites
Human and Animal Cells 610B1(HPS0331)