GSM4910646: GROseq.ctrl.st5.B_rep1; Drosophila melanogaster; OTHER
The GRO-seq protocol was adapted from Quarato et al., 2020 with modifications in the nuclei extraction protocol as follows: Embryos were resuspended in 1.5 ml nuclei extraction buffer (3 mM CaCl2, 2 mM MgCl2, 10 mM Tris HCl pH 7.5, 0.25 % Np40, 10 % Glycerol, Protease inhibitors and RNase inhibitor 4U/ml) and stroked 60 times. The lysate was centrifuged at 100g and nuclei were pellet at 1000g, and washed afterwards four times with nuclei extraction buffer. Nuclei were washed once with freezing buffer (50 mM Tris HCl pH 8, 5 mM MgCl2, 0.1 mM EDTA) and resuspended in 100 μl of freezing buffer. The Nuclear Run-On (NRO) reaction was performed by addition of 100 µl NRO 2x buffer (10 mM Tris HCl, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 1 % Sarkosyl, 0.5 mM ATP, CTP and GTP and 0.8 U/µl RNase inhibitor), using 1 mM Bio-11-UTP final concentration, follow by an incubation of 5 min at 30°C. RNA extraction was performed following the manufacturer's instructions with TRIzol reagent. Purified RNA was fragmented by the addition of reverse transcriptase buffer and 7 minutes incubation at 95°C. Biotinylated nascent RNAs were bound to 30 µl Dynabeads MyOne Streptavidin C1 (Invitrogen), washed and purified. Fragmented RNAs were repaired incubating with Polynucleotide kinase (Thermo scientific) for 30 minutes at 37˚C. RNA was purified with Phenol:Chloroform and precipitated. RNA was then ligated to 3' end adapter using T4 RNA ligase 2 Truncated KQ (home-made) for 16h at 15˚C. After ligation RNA was purified using SPRI beads and biotinylated RNA was enriched as described above. Purified RNA was ligated at 5' end using T4 RNA ligase1 for 2 h at 25˚C. Then, RNA was purified using SPRI beads and biotinylated RNA was enriched for a third time as described above. Purified RNA was reverse transcribed using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) following manufacturer conditions except that reaction was incubated for 1h at 50˚C. cDNA was PCR amplified with specific primers using Phusion High fidelity PCR master mix 2x (New England Biolab) for 18-20 cycles.