Ribo-seq of drosophila melanogaster: male head
Before the experiment, lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Triton x-100, 2 mM DTT, 100 U/ml SuperaseIn, 0.5 tablets of proteinase inhibitor, 100 g/ml emetine, and 50 M GMP-PNP) was prepared. Embryos of different stages were resuspended respectively in 1.5-2 ml cold lysis buffer in a cold room of 4 C, and homogenized on ice in a pre-chilled dounce homogenizer. Lysates were transferred to pre-chilled 1.5 ml RNase-free tubes, and clarified by spinning 10min at 2000 g at 4 C. Avoiding the fat layers and the precipitate, the supernatant was pipetted into a new pre-chilled tube, and stored at 80C. To enrich monosomes by sucrose density gradient centrifugation, 10-45% sucrose gradients were prepared in sucrose gradient buffer (250 mM NaCl, 15 mM MgCl2, 20 U/ml SuperaseIn, 20 g/ml emetine) using a Gradient Master (Biocomp Instruments, Canada) in polyclear centrifuge tubes. For ribosome profiling assay, lysates had been prepared were quantitated on a NanoDrop spectro-photometer to A260 ~1.5. For each sample, 200ul lysate was diluted 2:1 in digestion buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 15 mM CaCl, 1% Triton x-100, 1 mM DTT, 20 U/ml SuperaseIn, 20 g/ml emetine, 50 M GMP-PNP), with 3 U micrococcal nuclease per g of RNA added, and digested for 40 min at 25C. Reaction was quenched by adding EGTA to a final concentration of 6.25 mM. Up to 300 l of each sample was applied to the top of each sucrose gradient. Gradients were resolved by spinning for 3 hr at 35 000rpm at 4C in an SW-41 rotor (Beckmann Coulter), and fractionated using the Gradient profiler (Biocomp Instruments, Canada). When appropriate, monosome fractions were collected, flash-frozen in liquid nitrogen, and stored at 80C for subsequent library construction. Monosomes had been collected were digested with protease K and 10% SDS, incubated at 42C for 30min. The ribosome protected mRNA fragments were released, and extracted using acid phenol and chloroform. RNA was precipitated with alcohol for at least 3 hr at 80C, dissolved with 15 ul DEPC-treated water, and resolved on a 15% TBE-urea gel (Invitrogen by Life Technologies). A gel slab spanning 2832 nt (as measured by synthetic oligoribonucleotide size standards in a neighboring lane) was excised from the gel, eluted, and precipitated. Samples were depleted of rRNA by using Ribo-zero kit (Epicentre), dephosphorylated by PNK reaction (New England Biolabs) without ATP, and ligated to 3 adapter, and purified on a 15% TBE-Urea gel. The purified RNAs were phosphorylated by PNK reaction (New England Biolabs), and ligated to 5 adapter, subjected to reverse-transcription to cDNA using SuperScript III (Invitrogen). cDNAs were then further treated with synthesized biotinylated-oligos on DynaBeads (for further depletion of rRNA-derived products). cDNAs were amplified by 14 PCR cycles using Phusion DNA polymerase (NEB) and the products within the correct size ranges were purified from 20% TBE gels for quality tests (Fragment Analyzer, Agilent Technologies) and sequencing (Illumina HiSeq-2500 sequencer; run type: single-end; read length: 50 nt).