Design: 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 μg/ml emetine, and 50 μM GMP-PNP) was prepared. For larvae, pupae, and adults (males and females were separated, heads and bodies were divided, respectively), samples were grinded in cold room with pre-cooled pestle and molar containing liquid. The tissue powder was then transferred into 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 −80°C. 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 μg/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 μg/ml emetine, 50 μM GMP-PNP), with 3 U micrococcal nuclease per μg of RNA added, and digested for 40 min at 25°C. Reaction was quenched by adding EGTA to a final concentration of 6.25 mM. Up to 300 μl of each sample was applied to the top of each sucrose gradient. Gradients were resolved by spinning for 3 hr at 35 000rpm at 4°C 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 −80°C for subsequent library construction. Monosomes had been collected were digested with protease K and 10% SDS, incubated at 42°C 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 −80°C, dissolved with 15 ul DEPC-treated water, and resolved on a 15% TBE-urea gel (Invitrogen by Life Technologies). A gel slab spanning 28–32 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).
Submitted by: Peking University
Study:
study of translational regulation in Drosophila melanogastershow Abstracthide AbstractIn order to study translational control by cis-regulatory elements in untranslated region of mRNA, ribosome profiling assays during different developmental stages of Drosophila melanogaster and in S2 cells cultured in normal and stressful conditions were performed.
Library:
Name: larvae_ribo
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Runs:
1 run, 40.1M spots, 2G bases, 1.4Gb