GSM3323989: KD1_2; Drosophila melanogaster; OTHER
Nascent RNA isolation was performed as described at Weber et al and cap selection was done using GFP-elF4E recombinant protein generously provided by Dr. G. Zentner, Indiana University. RNA:GFP-elF4E complexes were pulled by GFP-nAb Magnetic Agarose beads (Allele Biotech) according to manufacturer's protocol. Sequencing library preparation for nascent RNA samples was performed according Illumina Protocol with slight modification.  After 3'- SR adaptor ligation, RNA was fragmented to 30-100 nucleotides with the RNA Fragmentation Reagent (Albion, AM8740) and purified according to Mayer et al, 2015. In order to prevent RNA fragments ligation, we performed 3'-OH phosphorylation with T4 PNK (3' phosphatase minus) before hybridization of reverse transcription primer and 5'-SR adaptor ligation. After reverse transcription with Superscript Reverse Transcriptase (Invitrogen) we purified cDNA on 15% polyacrylamide TBE-urea gel to avoid primer dimer formation, excised gel region between 50 and 300 nt and extracted cDNA. After PCR amplification we performed quality control (QC) and size selection. To be confident on PCR products we did cloning and sequencing by using Zero Blunt TOPO PCR Cloning kit (ThermoFisher Scientific). Positive colonies were harvested, plasmid DNA was purified with QIAprep Spin Miniprep kit (Qiagen) and digested with EcoR1.  Digested products were visualized on 1% agarose gel and sent to sequence (Eurofins Scientific).
GEO Accession
GSM3323989