Naked mole rat hypothalamus chromatin state map
Naked mole rat hypothalamus chromatin state map
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. The hypothalamus for eighteen NMR subordinates (14 female, 4 male) were collected for ChIP-sequencing using the same approach as in Hi-C sequencing. For ChIP-seq of CTCF, we collected the whole brain of two NMRs. Tissue was dounced in freshly prepared solution A ( 1% formaldehyde, 50mM Hepes-KOH, 100mM NaCl, 1mM EDTA, 0.5mM EGTA) and incubated for 20 mins at room temperature. The formaldehyde was then quenched with 1/20 volume of 2.5M glycine and incubated for 5 min. Tissue was then rinsed with ice cold PBS twice and centrifuge at 4 C at 2500 x rcf for 5 min. Crosslinked tissue was then lysed using Chromatin Easy Shear Low SDS Kit (Diagenode #C01020013). Chromatin was sheared into 100–500 bp DNA fragments by sonication (Diagenode Bioruptor Pico) for 8 cycles of 30s ON and 30s OFF. Approximately 1.5% of chromatin was used for input DNA extraction For each ChIP, chromatin lysates  were combined with 10ug of anti-H3K27ac (Active Motif #39133), anti-H3K4me2 (Millipore # 07-030), H3K27me3 (Millipore #07-449), H3K36me3 (Abcam #ab9050) and H3K9me2 (Abcam #ab8898) antibodies and incubated overnight rotating at 4C. For library preparation, all the ChIP DNA and input DNA were mixed with reagents from NEBNext Ultra II Library Prep Kit (NEBNext #E7645S) according to the manufacturer's protocol.This DNA was then amplified with barcoded primers for Illumina sequencing (NEBNext® Multiplex Oligos for Illumina® #E7335S). PCR amplifications were carried out as [98 °C 30 s, (98 °C 10 s, 65 °C 75s) ×9cycles, 65 °C 5 min, 4 °C hold].