GSM6595531: IgG_rep2; Mus musculus; RIP-Seq
Cells were lysed on ice using 50 μl wash buffer for 10 min. The preparation of protein A/G beads observed the instructions of the manufacturer, and their coupling with either anti-eIF4E or -eIF4E1B antibody took place. The lysate of oocytes was pre-treated with RQ1 DNase and incubated with antibody-coated beads. After extensive wash steps, the immunoprecipitated RNAs were fragmented, and experienced 3'-dephosphorylation and linker ligation, followed by reverse transcription on-beads. Then, first-strand cDNAs were derived from protein A/G beads and further captured by streptavidin C1 beads for pre-PCR and poly(A)-tailing to produce dsDNA as the template for IVT. IVT products were purified by the removal of DNA template using Turbo DNase and further purified by Agencourt RNA Clean beads based on the instructions of the manufacturer. Then, linear amplified RNA was transformed into cDNA and amplified by PCR with P7 and barcoded P5 index primers. Final PCR products with a size of 130-300 bp were excised from a 2% agarose gel and purified by use of Qiagen gel purification kit as per the instructions of the manufacturer. Illumina HiSeq 2500 at BerryGenomics was used for the single-end sequencing of all LACE-seq libraries.