GSM4728004: CJ_CG84-21_CoIP_WT_R2; Campylobacter jejuni subsp. jejuni CG8421; RIP-Seq
Cas9-bound RNAs (eluted RNA) were extracted from beads harboring anti-FLAG immunoprecipitated proteins. For both, untagged and Cas9-3xFLAG co-immunoprecipitated samples, RNA was purified from the bound complexes using Phenol:Chloroform:Isoamylalcohol extraction. Eluted RNA was precipitated overnight at -20°C. Eluted RNA from co-immunoprecipitation experiments was used as such. At first, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. RIP-seq library prepared using adapter ligation protocol.