GSM6454906: wt naive  PRO-seq Replicate 1; Drosophila melanogaster; OTHER
Precision Run-On sequencing (PRO-seq) and the more sensitive qPRO-seq were performed according to the papers 'Kwak, H., Fuda, N.J., Core, L.J., and Lis, J.T. (2013). Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950-953.' and 'Judd, J., Wojenski, L.A., Wainman, L.M., Tippens, N.D., Rice, E.J., Dziubek, A., Villafano, G.J., Wissink, E.M., Versluis, P., Bagepalli, L., et al. (2020). A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq). bioRxiv, 2020.2005.2018.102277.'. 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 u/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 mesh followed by douncing with a tight pestle for 10 strokes. Nuclei were pelleted at 700 g 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, 5 mM DTT). For PRO-seq, isolated nuclei corresponding to approximately 10 million cells, and for qPRO-seq from 1 million cells, were resuspended in buffer D and stored at -80°C for subsequent nuclear run-on (NRO) reactions with biotinylated nucleotides. Library construction was performed according to the papers  'Kwak, H., Fuda, N.J., Core, L.J., and Lis, J.T. (2013). Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950-953.' and 'Judd, J., Wojenski, L.A., Wainman, L.M., Tippens, N.D., Rice, E.J., Dziubek, A., Villafano, G.J., Wissink, E.M., Versluis, P., Bagepalli, L., et al. (2020). A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq). bioRxiv, 2020.2005.2018.102277.'. Samples were sequenced single-end (1x75 bp) on the Illumina NextSeq 550 platform at the BEA core facility, Karolinska Institutet, Stockholm.