Dmel_female_control_ribo
Ribosome protected fragments were released from monosomes collected by sucrose gradiednt centrifugaion, with Proteinase K (Sigma-Aldrich) and 10% SDS at 42 °C for 30 min. Then the released mRNAs were purified on a 15% TBE-Urea gel (selecting 28-32nt fragments), with rRNA removed with ribo-zero rRNA removal kit (illumina), dephosphorylated by PNK reaction (New England Biolabs), ligated with 3’-adaptor and purified on a 15% TBE-Urea gel. The purified RNAs were phosphorylated by PNK reaction (New England Biolabs), ligated to 5’ adapter, and subjected to reverse-transcription into cDNAs. The cDNAs were treated with synthesized biotinylated oligos on DynaBeads for further depletion of the rRNA-derived products and subjected to PCR and gel purification, and sequenced on Illumina Hiseq platform and single-end reads were generated