GSM9136899: DDX5 K144N mutant Replicate #1; Homo sapiens; RIP-Seq
Enhanced CrossLinking and ImmunoPrecipitation (eCLIP) was performed by Eclipse BioInnovations following the published protocol (PMID: 27018577) without modifications. HepaRG cells expressing doxycycline-inducible FLAG-tagged DDX5 (wild-type or ATPase/RNA-binding deficient K144N mutant) were UV crosslinked, and protein-RNA complexes were immunoprecipitated using anti-FLAG antibodies. Following SDS-PAGE and transfer to PVDF membranes, regions corresponding to 69–144 kDa were excised to isolate crosslinked RNA. Size-matched input (SMInput) controls were processed in parallel. RNA was visualized via chemiluminescence, and libraries were constructed from immunoprecipitated and input RNA using reverse transcription and PCR amplification (8–11 cycles). Final libraries ranged from 225–252 nt in average size, with yields of 966–2763 fmol, and quality was assessed via Agilent TapeStation. Four immunoprecipitation and four input libraries were prepared for downstream sequencing and analysis. Enhanced CrossLinking and ImmunoPrecipitation (eCLIP) was performed by Eclipse BioInnovations following the published protocol (PMID: 27018577) without modifications. HepaRG cells expressing doxycycline-inducible FLAG-tagged DDX5 (wild-type or ATPase/RNA-binding deficient K144N mutant) were UV crosslinked, and protein-RNA complexes were immunoprecipitated using anti-FLAG antibodies. Following SDS-PAGE and transfer to PVDF membranes, regions corresponding to 69–144 kDa were excised to isolate crosslinked RNA. Size-matched input (SMInput) controls were processed in parallel. RNA was visualized via chemiluminescence, and libraries were constructed from immunoprecipitated and input RNA using reverse transcription and PCR amplification (8–11 cycles). Final libraries ranged from 225–252 nt in average size, with yields of 966–2763 fmol, and quality was assessed via Agilent TapeStation. Four immunoprecipitation and four input libraries were prepared for downstream sequencing and analysis.