title: Illumina HiSeq 2500 paired end sequencing; Chromatin accessibility in mouse hypothalamic arcuate nucleus in puberty onset
design: Chromatin accessibility in mouse hypothalamic arcuate nucleus in puberty onset
library_construction_protocol: 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. Freshly harvested ARC tissues (1x10^5) washed by cold PBS were ground in liquid nitrogen, then filtered by 40 μm cell strainer (Corning, New York City, NY, USA), and centrifuged with 12000 g at 4 oC for 10 min.The supernatant was transferred to another new Eppendorf tube, and incubated with 50 μl cold RSB buffer (10 mM NaCl, 3 mM MgCl 2, 10 mM Tris-HCl, pH 7.5) containing with 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin on ice for 3 min, and added additional 1 ml cold RSB buffer containing 0.1% Tween-20, and centrifuged with 500 g at 4 oC for 5 min, and discarded the supernatant. The precipitation was incubated with 50 μl transposition mix (Vazyme, Nanjing, China) at 37 oC for 1000 rpm rotation. Transposed DNA of the mixture was isolated by Qiagen MinElute PCR Purification Kit (Qiagen, Hilden, Germany), and amplified by the appropriate PCR condition.