title: GSM6468246: Toll10B 2to4h CUT&Tag Rpb3 Replicate 2; Drosophila melanogaster; OTHER
library_construction_protocol: Nuclear extraction was performed as described by 'Hainer, S.J., and Fazzio, T.G. (2019). High-Resolution Chromatin Profiling Using CUT&RUN. Curr Protoc Mol Biol 126, e85.' and CUT&TAG was performed essentially as described by 'Kaya-Okur, H.S., Wu, S.J., Codomo, C.A., Pledger, E.S., Bryson, T.D., Henikoff, J.G., Ahmad, K., and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930.'. Nuclear extracts were prepared using a glass douncer and loose pestle 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)) and centrifuged at 700 g for 10 min at 4°C. The nuclear pellets were resuspended in Nuclear Extraction buffer. Nuclei corresponding to 50 embryos per reaction (2-4 h AEL), 100 embryos (1.5-2 h AEL) or 200 embryos (60-80 min AEL) were incubated with 30 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. Nuclei-bead complexes were resuspended in 100 μl Antibody buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 2 mM EDTA pH 8.0 and 0.1% BSA supplemented with protease inhibitor cocktail (Roche)). For Toll mutant CUT&Tag, 1 μl of rabbit anti-BRD4/fs(1)h, rabbit anti-CycT, rabbit anti-Cdk9, rabbit anti-Rpb3 (a kind gift of John Lis), rabbit anti-RNA Polymerase II CTD repeat YSPTSPS (phosphor S5) (5SerP) (Abcam, ab5131) and rabbit anti-RNA Polymerase II CTD repeat YSPTSPS (phospho S2) (2SerP) (Abcam, ab5095) overnight at 4°C. For yw; PCNA-eGFP CUT&Tag, 1 μl of rabbit anti-BRD4/fs(1)h, rabbit anti-Cdk9 and rabbit anti-CBP. Following overnight incubation, tagmentation was performed using 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: 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 13 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). Libraries were pair-end (2 x 37 bp) sequenced on an Illumina NextSeq 550 platform at the BEA core facility, Karolinska Institutet, Stockholm.