title: GSM4997985: S2 Rbp9 WT; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Total RNAs were extracted from S2R+ cells transfected as described above using Trizol. Total RNA-seq libraries of L1CNS samples were prepared using TruSeq Stranded Total RNA Library Preparation Kit (Illumina) with 2 ug total RNA as starting materials for each sample. BrdU-CLIP libraries were made using S2R+ cells with overexpression of wildtype Elav, Rbp9, Fne and their RRM3XMT counterparts based on published with some optimizations. Protein-RNA complexes are immune-precipitated using anti-FLAG antibody (Sigma Aldrich) followed by stringent washes and ligation of the 3' linker. After digestion of the protein, the RNA is purified and reverse transcribed using a dNTP mix where dTTP is replaced with BrdUTP. The RT primer has 5' and 3' anchor sequences used later for PCR amplification separated by an APE1 cleavage site. The BrdUTP incorporated into the resulting cDNA allows on-bead cDNA purification using anti-BrdU antibody (Sigma Aldrich). The purified cDNA is then circularized and re-linearized on-bead using CircLigase II and APE1, respectively, which places the two PCR anchors in the correct orientation.