Standard NextSeq 2000 single-end Sequencing; PRO-seq data from SPT5-HA-iLEXY embryos at different developmental time windows and after different blue-light depletion conditions
PRO-seq data from SPT5-HA-iLEXY embryos at different developmental time windows and after different blue-light depletion conditions
The following line was used generate the CRISPR-Cas9 mediated knock-in of the iLEXYs tag to the spt5 locus: w[1118]; Pbac{y[+mDint2] = vas-Cas9}VK00027 (vasa-Cas9) (Bloomington stock 51324). Drosophila melanogaster (D. melanogaster) and Drosophila virilis (D. virilis) were maintained in a standard cornmeal-agar medium and grown at 25°C. The spt5-HA-iLEXYs line was always maintained under safelight conditions. ; For CUT and tag, embryos at the appropriate stage and after blue light illumination as described in the methods/results were dechorionated, and fixed in 1.8% formaldehyde (Sigma-Aldrich N.1.04003.1000) in cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, pH 8) for 15 min.  Formaldehyde was quenched with 125 mM Glycine (Millipore N.1.04201.1000) in PBT (PBS with 0.1% Triton-X100).  After two washes in PBT, embryos were dried on a Nitex membrane (Sefar N.03-125/45), snap-frozen in liquid nitrogen and stored at -80°C until further use.  For RNA-seq and PRO-seq, embryos at the appropriate stage and after blue light illumination as described in the methods/results were dechorionated and snap-frozen in liquid nitrogen and stored at -80°C. ; The protocol for isolation of transcriptionally active non-crosslinked nuclei from embryos for PRO-seq experiments. The protocol is an adaptation of the procedure from Hunt et al., 2024 (PMID: 38166964). Please refer to manuscript for details. ; PRO-seq protocol generally applicable to various systems, this version is modified for Drosophila embryonic nuclei with spike-in mammalian control (Judd et al. 2020; 10.1101/2020.05.18.102277). Please refer to manuscript for details.