GSM9014027: H3K27ac_BrainStem_KO; Mus musculus; OTHER
For the pituitary and spinal cord, subsequent procedures were directly performed after samples were removed. The bone marrow was isolated from femur and tibia bones. Muscles were removed and both ends of bones were carefully trimmed. The bone marrow cavity was flushed 2-3 times with DPBS to collect most of the cells. Tubes were then centrifuged at 300 g for 5 min at 4°C to collect cell pellets. For the stomach, intestine, and bladder, their luminal content was cleaned up before frozen in dry ice. Other tissues were directly frozen once removed. Frozen tissues were ground in liquid nitrogen. The ground powder was used for subsequent library construction and can be stored at -80°C. Concanavalin A (ConA) coated magnetic beads were freshly activated with Ca²⁺ and Mn²⁺. Nuclei were extracted and a total of 10,000 nuclei were incubated with activated ConA beads. Supernatant was next removed, and beads were incubated with antibody (1:50) on a rotator at 4°C overnight, followed by incubated with secondary antibody (1:100) for 1 hr at room temperature. Beads were washed three times and suspended in dig300-wash buffer pre-mixed with pG-Tn5 (1:100), incubated for 1 hr at room temperature. After washing beads for three times, tag buffer was added to each well, then incubated on a rotator at 37 °C for 1 hr. After centrifugation, beads were washed once and resuspended. Lysis buffer was added to each well, and incubated for 2 hr at 65 °C. After adding 10% Tween20, TruePrep CUT&Tag Amplification Mix (Vazyme), 10 mM MGI-P5, and 10 mM MGI-P7 primer were added. Then PCR program was performed. Finally, 1.5 × VAHTS DNA Clean Beads were used to purify the library.