GSM7754587: Methyl_seq_6B2; Mus musculus; Bisulfite-Seq
At 3 month age, mice were euthanized and hypothalamus was flash frozen on dry ice for DNA extraction. Extracted DNA was used to perform whole epigenome methylation sequencing. Methyl-Seq library preparation was performed using the SureSelectXT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing (Agilent Technologies, USA) as described by the manufacturer's protocol. Briefly, 3 ìg of DNA was sheared to fragments of 100-200 bp using a Covaris M220 instrument followed by end repair and adapter ligation. DNA fragments were then subjected to hybridization to the SureSelectXT Methyl-Seq Mouse Capture Library for 16 h (Agilent Technologies, USA). Hybridized products were purified by capture with streptavidin beads and then subjected to bisulfite conversion using the Zymo EZ-DNA Methylation-Gold kit (Zymo Resesarch, USA). Libraries were enriched by PCR amplification. Unique indexes were then ligated to the amplified libraries by PCR amplification. The quality of the libraries was determined after end repair, adapter ligation, and indexing by Agilent's Bioanalyzer 2100 using a high sensitivity DNA kit, to verify that the library peak size fell within the range recommended by the manufacturer's protocol. Quantification of the final libraries was performed using the Qubit dsDNA high sensitivity assay (ThermoFisher Scientific, USA). Sequencing was performed on Illumina's NextSeq 500 instrument with pair-end 100 bp reads.