GSM8457916: PHX3174, control_IgG, eCLIP; Caenorhabditis elegans; RIP-Seq
Five 10 cm dishes of C. elegans were UV crosslinked at 400 mJoules/cm2 with 254 nm radiation and snap frozen. C. elegans was then lysed and treated with RNase I to fragment RNAs. Sigma monoclonal anti-FLAG M2 antibody (cat # F1804-1MG)or Sheep Anti-Mouse IgG (ThermoFisher Scientific, cat# 11202D) was pre-coupled to Dynabeads M-280, added to the lysate, and incubated overnight at 4°C. Prior to immunoprecipitation, 2% of the sample was taken as the paired input sample, with the remainder being magnetically separated and washed with lysis buffer only. eCLIP was performed by excising the area from ~60 kDa to ~135 kDa, and RNA adapter ligation, IP-western blot, reverse transcription, DNA adapter ligation, and PCR amplification were performed.