Standard NextSeq 500 paired-end Sequencing; RNA-seq data from SPT5-HA-iLEXY embryos at different developmental time windows and after different blue-light depletion conditions
RNA-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. ; Embryos were homogenised in TRIzol LS (ThermoFisher Scientific N.10296028) with a pestle on ice. RNA was isolated using standard phenol-chloroform extraction, and the remaining DNA digested with RNase-free DNase I (Roche N.4716728001) for 30 min.  RNA quality was checked on a Bioanalyzer (Agilent).  For the depletion experiments at 3-4 h, RNA-seq was performed using the Ovation RNA-Seq System 1-16 for Model Organisms (from NuGEN, now Tecan Genomics) following the manufacturer's protocol.  For all other experiments, RNA-seq was performed from 1 μg of total RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB N.E7420) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB N.E7490) according to the manufacturer's instructions.  All RNA-seq experiments were performed in two biological replicates.  Final libraries were checked on a Bioanalyzer and multiplexed.