GSM4598692: control IP1; Drosophila melanogaster; RIP-Seq
Cell pellets were thawn on ice and iCLIP was performed as described in Buchbender et al. 2019. Briefly, native cell extracts were prepared and subjected to RNase treatment using 10U of RNase I (Ambion). Immunoprecipitation was performed with anti-FLAG antibody (M2, Sigma Aldrich) or control serum on Dynabeads Protein A (Life Technologies) for 2h at 4°C. After washing four times with washing buffer (50mM Tris/Cl pH7.4, 1M NaCl, 0.05% Tween 20), the precipitated RNA-protein complexes were eluted. The co-immunoprecipited RNA was dephosphorylated, ligated to a 3'-RNA linker and 5'-radiolabeled with T4 PNK and [γ-³²P]-ATP. Samples were subjected to neutral SDS-PAGE (NuPAGE, Invitrogen) and transferred to a nitrocellulose membrane. Protein/RNA-complexes were visualized by autoradiography. Mei-P26:RNA-complexes were cut from the membrane, proteins were digested with Proteinase K and RNA was subjected to iCLIP library preparation. Library preparation occurred as previously described by Buchbender et al., 2019. In brief, after immunoprecipitation, 3' end dephosphorylation occurred, followed by linker ligation to the 3' end of the RNA (see above). After further purification of the ribonucleoprotein complexes and removal of the protein moiety by proteolytic digestion (as described above), reverse transcription occurred using a primer directed against the linker sequence. Purification of the cDNAs occurred via MyONE resin, followed by ligation of an adapter to the 3' end of the cDNA and a second clean-up step employing MyONE resin. 6 cycles of PCR were performed to pre-amplify the library. After size selection via ProNex beads, the final PCR amplification step occurred, introducing Illumina-compatible overhangs for sequencing followed by an additional round of ProNex purification. iCLIP2 protocol as described in Buchbender et al. 2019.