RNA-seq of drosophila melanogaster: 12-24h embryo
a total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Briefly, 3 μg Poly(A)+ RNA selected on oligo-dT25 DynaBeads and fragmented. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H, with dTTP replaced by dUTP. DNA fragments were ligated to 3’ adaptor, size selected, and purified with AMPure XP system (Beckman Coulter, Beverly, USA). Prepared cDNAs were amplified by PCR with Phusion High-Fidelity DNA polymerase, purified, and sequenced on Illumina Hiseq platform and single-end reads were generated.