GSM8509092: Embryo_CRY2CBP_Dark_pSer5_PolII_R1; Drosophila melanogaster; OTHER
Embryos were  dechorionated, rinsed in embryo wash buffer (PBS, 0.1% Triton X-100), and crude nuclear extracts prepared using a glass douncer in Nuclear Extraction buffer (20 mM HEPES pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol with protease inhibitor cocktail (Roche))90 and centrifuged at 700g for 10 min at 4°C. The nuclear pellets were resuspended in a Nuclear Extraction buffer and mixed with Drosophila virilis nuclei extracted from 3rd instar larvae (approximately 10% of the total number of nuclei). Nuclei corresponding to at least 100 embryos per reaction were incubated with 25 μl of BioMag®Plus Concanavalin A beads (Polysciences) (prepared in Binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1mM CaCl2, and 1 mM MnCl2)) on a nutator for 10 min at 4°C. CUT&Tag was performed according to a published protocol Kaya-Okur, H.S., Janssens, D.H., Henikoff, J.G., Ahmad, K., and Henikoff, S. (2020). Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc 15, 3264–3283. https://doi.org/10.1038/s41596-020-0373-x. We used the following antibodies: 1 μl rabbit H3K27ac (Abcam, ab4729), 1 μl rabbit H3K18ac (Abcam, ab1191), 1 μl rabbit H3K9ac (Abcam, ab4441), 1.2 μl rabbit CBP, 1 μl TBP (a kind gift from Jim Kadonaga, UCSD), 1.2 μl rabbit anti-BRD4/fs(1)h (a gift of Renato Paro, kindly provided by Nicola Iovino), 1.2 μl rabbit anti-CycT (a kind gift of Kazuko Hanyu-Nakamura), rabbit anti-RNA Polymerase II CTD repeat YSPTSPS (phospho-serine 5) (5SerP) (Abcam, ab5131), rabbit anti-RNA Polymerase II CTD repeat YSPTSPS (phospho-serine 2) (2SerP) (Abcam, ab5095). Following overnight incubation, the experimental procedure was followed using guinea pig α-rabbit antibody (Antibodies online cat. no. ABIN101961) secondary antibodies and purified pA-Tn5 (Protein Science Facility, KI, Stockholm). Tagmented DNA was PCR amplified using custom i5 and i7 PCR primers and Phusion® High-Fidelity PCR Master Mix with GC Buffer (NEB). PCR conditions were as follows: 72°C for 5 min, 98°C for 30 s, followed by thermocycling (98°C for 10 s and 63°C for 10 s) for 14 cycles and final extension at 72°C for 1 min.  Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter) (1.1:1 bead to sample volume ratio).