NextSeq 550 paired end sequencing; Transcriptome and DNA (hydro)methylation in mouse hypothalamic arcuate nucleus in puberty onset
Transcriptome and DNA (hydro)methylation in mouse hypothalamic arcuate nucleus in puberty onset
Mice were sacrificed via cervical dislocation, and the whole brains were isolated immediately. The hypothalamic ARC tissues were obtained according to the orientation of 0.4 mm lateral, 1.60 mm posterior to bregma, 7.40 mm below the surface of the dura Genomic DNA of ARC in two groups were extracted using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).  The 200 ng high-quality DNA was then digested by restriction endonucleases MspI (NEB) and subjected to 3′-end blunting and single nucleotide (A) addition and adaptor ligation. For RRHP, 5hmC positions at the adapter junctions were modified by T4 phage β-glucosyltransferase (NEB), and non glucosyl-5hmCs were removed by another round of MspI digestion. The 250-500 bp fragments were then selected and treated with bisulfite conversion using Epitect Bisulfite Kit (Qiagen) according to the manufacturer's instructions.