title: GSM7868254: Ribo-seq bam,hs-bam 0.5hr AHS rep2; Drosophila melanogaster; OTHER
library_construction_protocol: Ovaries were dissected for each sample in cold Dissection buffer (PBS 1X, 0.01% Tween 20, 100 μg/ml cycloheximide) and stored at -80°C. Frozen samples were homogenised with an electrical pestle in Polysome extraction buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 150 mM NaCl, 0.5% Triton x-100, 1 mM DTT, 100 μg/ml cycloheximide, protease inhibitor, 25 u/ml Turbo DNAse), then passed 20 times through a 26-gauge needle. The lysate was centrifuged at 20,000g for 10 min at 4°C to pellet tissue debris. 100-140 μg of total RNA was used for Ribo-seq. Total RNA was treated with 1.25 units of RNase I (Ambion) per μg RNA, and the quenched with SuperaseIn (0.8 units per μg RNA). The sample was brought to 1 ml by adding Polysome extraction buffer and subjected to a 3 ml 34% sucrose cushion, by centrifugation at 70,000 rpm for 5.5 hr at 4°C. The resulting pellet was resuspended in nuclease free water with 1% SDS. RNA was extracted with hot phenol-chloroform and purified by Zymo RNA Clean & Concentrator kit. rRNA was depleted as for the RNA-seq through binding of complementary oligos and treatment with RNase H. Size selection was performed on a 15% Urea PAGE Gel, cutting the band corresponding to 28 to 34 bp. RNA was purified by ZYMO small-RNA PAGE recovery kit. RNA was treated with 1 ul of T4 PNK for end repair and libraries were produced with the NEBNext Small RNA Library Kit, according to manufacturer9s instructions. Libraries were size selected at 148-154 nucleotides, corresponding to ligated constructs from 28 to 34 nt RNA fragments.