Standard Element AVITI paired-end Sequencing; DFF-seq data from WT embryos at different developmental time windows
DFF-seq data from WT embryos at different developmental time windows
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. ; The protocol for isolation of transcriptionally active non-crosslinked nuclei from embryos for PRO-seq and DFF-seq experiments. The protocol is an adaptation of the procedure from Hunt et al., 2024 (PMID: 38166964). Please refer to manuscript for details. ; DFF-seq protocol used to determine nucleosome positioning at 2-3, 3-4, and 18-20 h wild-type embryos. Between 2 and 4 million nuclei were used in each DFF digestion reaction. 50 uL of nuclei suspension were thawed and mixed with 150 uL of 1x T4 DNA ligase buffer (NEB B0202SVIAL) supplemented with 0.1 mg/mL BSA and 1x Roche cOmplete Protease inhibitors (cat.N. 11836170001). ~1 ug of activated DFF40 was added and the reaction incubated for 20 min at 37°C on a thermoshacker. 20 mM EDTA (final concentration) and 0.5% SDS were added to stop the reaction. Samples were digested with proteinase K at 50°C for 2 hours and DNA purified by standard phenol/chloroform/ethanol extraction procedure. DNA digestion down to mononucleosomal fragments (150-160 bp) was confirmed on agarose gel and NGS libraries were prepared for sequencing from 50 ng of digested material using NEBNext Ultra II library prep kit (NEB E7645S). The PCR products were purified with 1.2x Agencourt AMPure XP beads (Beckman Coulter, N.A63881), quantified with Qubit, run on the Bioanalyzer with HS DNA reagents, and sequenced at the EMBL Genomics Core Facility.