Ribosomal Profiling of Drosophila melanogaster Oregon-R Whole Embryos
Embryos from each time period (i.e., sample) were treated with harringtonin in mild lysis buffer followed by the addition of cycloheximide and immediate grinding. Samples were subjected to a 10%-50% sucrose gradient and all polysomes were collected and combined. Collected polysome fractions were pelleted via sucrose cushion (34%) and subjected to RNaseI digestion. After resuspension another cushion (34%) was performed and RNA was coprecipitated with GlycoBlue (Thermo Fisher, cat. No. AM95150). Recovered RNA was then run on a 15% TBE-Urea Gel (Thermo Fisher cat. no. EC68852BOX), followed by gel size selection to isolate ribosome protected fragments (26-31 nt) (Zymo Research, cat. no. R1070). Following end-repair phosphorylation of RNA molecules, libraries were constructed with the NEBNext Small RNA Library Preparation Kit according to manufacturers recommendations (NEB, cat. no. E7330).