GSM8508885: Embryo_CRY2CBP_Dark_PROseq_R1; Drosophila melanogaster; OTHER
A variant of PRO-seq, qPRO-seq was performed on CBPdeGrad and CBPCRY2 embryos collected for one hour and aged for a further two hours (2–3 h AEL). Collected embryos were dechorionated in dilute bleach and rinsed thoroughly in embryo wash buffer (PBS, 0.1% Triton X-100) before being flash-frozen in liquid nitrogen and stored at −80°C. qPRO-seq was performed as previously described Judd, J., et al., 2020, bioRxiv, https://doi.org/10.1101/2020.05.18.102277. Briefly, embryos were resuspended in cold nuclear extraction buffer A (10 mM Tris-HCl pH 7.5, 300 mM sucrose, 10 mM NaCl, 3 mM CaCl2, 2 mM MgCl2, 0.1% Triton X, 0.5 mM DTT, protease inhibitor cocktail (Roche) and 4 µ/ml RNase inhibitor (SUPERaseIN, Ambion)), transferred to a dounce homogenizer and dounced with the loose pestle for 20 strokes. To remove large debris, the suspension was passed through Miracloth tissue (Merck, 475855-1R) followed by douncing with a tight pestle for 10 strokes. Nuclei were pelleted at 700g for 10 min at 4°C and washed twice in buffer A and once in buffer D (10 mM Tris-HCl pH 8, 25% glycerol, 5mM MgAc2, 0.1 mM EDTA, 0.5 mM DTT). For qPRO-seq, we used 2 million nuclei resuspended in buffer D and stored at −80°C. For further spike-in normalization, they were mixed with Drosophila virilis embryo nuclei (2% of the total number of nuclei). Nuclear run-on assays were performed in biological duplicatesusing all four biotinylated dNTPs  exactly as previously described  in Judd, J., et al., 2020, bioRxiv, https://doi.org/10.1101/2020.05.18.102277. Libraries were produced as descibed in Judd, J., et al., 2020, bioRxiv, https://doi.org/10.1101/2020.05.18.102277. We have skipped preCR step and performed amplification with 15 cycles based on test amplification. Libraries were PAGE purified before sequencing.