title: GSM9180761: Anopheles_stephensi_ovaries_sRNA_rep1; Anopheles stephensi; ncRNA-Seq
library_construction_protocol: Small RNA sequencing was performed on ovaries and eggs collected from a variety of insects. About 10 pairs of ovaries from 1 week to up to 3 months old females and eggs within up to 8 hours of egg laying have been collected per replicate. Total RNA was extracted by TRIZOL (Thermo Fisher) and the genomic DNA was removed by DNase I (Thermo Fisher). Small RNAs were first size-selected from the total RNA by 12% w/v UREA-PAGE using radio-labelled 19mer (5′-CGUACGCGGGUUUAAACGA) and 35mer (5'-CUCAUCUUGGUCGUACGCGGAAUAGUUUAAACUGU) spikes, and oxidised using sodium periodate. The oxidised small RNAs were further size-selected by PAGE. The size-selected oxidised small RNAs were ligated to the 3' adapter 5rApp/NNNNAGATCGGAAGAGCACACGTCT/3ddC, and then to the 5' adapter ACACUCUUUCCCUACACGACGCUCUUCCGAUCUNNNN where Ns are randomised. The first strand cDNA synthesis was performed using SuperScript II (Thermo Fisher) and the RT primer GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT. The library was amplified using KAPA LongRange DNA polymerase (Sigma) with a universal TruSeq forward primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and a barcode-containing TruSeq reverse primer (e.g. CAAGCAGAAGACGGCATACGAGATxxxxxxGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT where xxxxxx were the barcode).