Chromatin accessibility in the adult male and female naked mole rat hypothalamus before and during colony removal
Chromatin accessibility in the adult male and female naked mole rat hypothalamus before and during colony removal
Naked mole-rats were bred in-house and maintained in stable colonies (i.e. established breeders were present) housed in large (45.75 cm L × 24 cm W × 15.25 cm H) and small (30 cm L × 18 cm W × 13 cm H) polycarbonate cages connected by plastic tubes (25 cm L × 5 cm D). Animals were fed sweet potato ad libitum supplemented with wet 19% protein mash (Envigo RMS, Inc.) and kept on a 12:12 light/dark cycle at 28–30 C. All procedures were approved by the University Animal Care Committee and performed in accordance with federal and institutional guidelines.  Twelve NMR hypothalamus samples between two and four years of age were collected using the same method as in Hi-C and ChIP-sequencing. These twelve NMRs were divided into subordinates (SUB), and subordinates were removed from the colony for one week (EXSUB-wk1), in both males, and females (N=3 per developmental stage/sex). NMR hypothalamus were dissected, flash frozen and stored at -80C for up to four months.   Tissue was homogenised in a dounce homogenizer in PBS, then washed 2x with ice-cold PBS. Cells were counted with a CountessTM using 1:1 dilution of Trypan Blue. ATAC-Seq was performed according to Buenrostro, et al. (2015). Briefly, 100000 cells were used for each sample and resuspended in 50uL of lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Cells were spun for 10 mins at 600xg and 4C to prepare a crude nuclei extract. Nuclear pellets were then resuspended in transposition mix (25uL 2X TD buffer, 2.5uL TD [Illumina #20034198], and 23uL nuclease-free water) and incubated in a thermomixer at 37C for 30 mins shaking at 800rpm. DNA was isolated using the DNA Clean & Concentrator-5 kit (Zymo  #D4013) and eluted in 11.5uL of 10mM Tris-HCl, pH 8.0.  Libraries were prepared using 10uL of each sample. To generate libraries 50uL PCR reactions were made using 25uL NEBNext® High-Fidelity 2X PCR Master Mix(NEB #M0541L) , 2.5uL of universal Ad1_noMx primer, 2.5uL of Ad2.x barcoded primer and 10uL nuclease-free water. Reactions were run according to recommended conditions with an annealing temperature of 63C and amplified for a total of 5 cycles. qPCR was subsequently run using 5uL of each 50uL PCR reaction to determine the number of additional cycles choosing a cycle number which provided ⅓ maximum fluorescence value. After subsequent PCR cycles, libraries were cleaned using a two-sided AMPureXP bead clean up using 0.5X then 1.3X beads. Libraries were eluted in a final volume of 21uL of 10mM Tris-HCl, pH 8.0, and quantified with the DNA-HS Qubit assay.