SUB_Female_Hypothalamus_HiC_Rep2_p
Hi-C 20 subordinate (wild-type) female naked mole-rats between 1-4 years of age were anesthetized with isoflurane and rapidly decapitated. The brain was sliced coronally into 1 mm sections on an acrylic block. Sections were transferred to cold PBS and the hypothalamus removed using a scalpel blade and flash frozen whole in isopentane on dry ice. Hi-C experiments were performed using the Arima Hi-C kit (Arima Genomics) according to the manufacturerâ€™s instructions. Briefly, pooled flash frozen brain tissues were cross-linked with 2% formaldehyde for 20 min at RT in buffer containing 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 50mM HEPES (pH8.0) in 1X PBS. DNA content was estimated and 3ug was used as input per biological replicate. Chromatin was digested with enzyme cocktail included in the kit. Ends were filled-in in the presence of biotinylated nucleotides, followed by subsequent ligation. Ligated DNA was sonicated using the Diagenode Picoruptor ( 10 Cycles of 30 Secs On/Off)  to an average fragment size of 400 bp DNA was then subjected to a double-size selection to retain DNA fragments between 200 and 600 bp using Ampure XP beads (Beckman Coulter). Biotin-ligated DNA was precipitated with streptavidin-coupled magnetic beads (included in the kit). The library was prepared using Swift Biosciences Accel-NGS 2s Plus library kit (Cat # 21024). Final amplification before sequencing was done with KAPA Library Amplification Kit ( Cat # KK2620).