GSM6190430: Mid-T-II; Drosophila melanogaster; OTHER
We purified mRNAs in polysomes, away from monosomes (80S), ribosomal subunits (40S, 60S). Footprinting was performed overnight at 4°C with RNaseI (Invitrogen), stopped with SUPERase·In TM RNase inhibitor (Invitrogen) and precipitated. mRNA from total cytoplasmic lysate, was purified using oligo (dT) Dynabeads (Invitrogen) and fragmented by alkaline hydrolysis. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols. NEBNext Small RNA Library Prep Set for Illumina (NEB, E7300), rRNA depleted using biotinylated DNA fragments and oligos from rRNAs and going through two consecutive rounds of subtractive hybridization using MyOne Streptavidin C1 Dynabeads (Invitrogen). After ethanol precipitation, samples were processed as NEB guidelines. We purified mRNAs in polysomes, away from monosomes (80S), ribosomal subunits (40S, 60S). Footprinting was performed overnight at 4°C with RNaseI (Invitrogen), stopped with SUPERase·In TM RNase inhibitor (Invitrogen) and precipitated. mRNA from total cytoplasmic lysate, was purified using oligo (dT) Dynabeads (Invitrogen) and fragmented by alkaline hydrolysis. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.