title: GSM8239049: Control 2; Mus musculus; Bisulfite-Seq
library_name: GSM8239049
library_construction_protocol: Genomic DNA was extracted using the Quick-gDNA MiniPrep kit (Zymo Research, Irvine, CA, USA, D3024) following the manufacturer's instructions for tissues. The steps of RRBS analysis involved DNA digestion using the restriction enzyme MspI, which cuts DNA at its recognition site, CCGG, independently of CpG methylation status. Subsequent reactions for DNA end repair, A-tailing and adaptor ligation were followed by Illumina sequencing (NextSeq 500) after size selection. Bisulfite conversion was then performed for total genomic DNA. Bisulfite conversion with sodium bisulfite allows conversion of cytosines to uracils in a single-stranded DNA, whereas 5-methylcytosines (5mCs) are resistant to this conversion and remain as cytosines, allowing 5mCs to be distinguished from unmethylated cytosines. During subsequent PCR amplification, the DNA sequences are amplified with primers specific for one strand of uracil converted from methylated cytosine, and uracil is amplified as thymine. If cytosine in the CpG dinucleotide exists as 5mC, PCR recognizes 5mC as cytosine.