GSM3669535: TT-Fam120a_Untagged_ES_iCLIP; Mus musculus; OTHER
mESCs were UV-crosslinked and harvested. Crosslinked cell pellets were resuspended in lysis buffer, partially digested with RNase I, and serially immunoprecipitated with FLAG and HA antibodies. Protein-RNA complexes were resolved on SDS-PAGE gels and transferred to nitrocellulose. Regions corresponding to crosslinked Fam120a complexes were extracted from nitrocellulose with proteinase K digestion and RNA isolated by phenol extraction. Dephosphorylated RNA was ligated to a 3' linker and reverse transcribed. The resulting cDNA was purified on a denaturing polyacrylamide gel, circularized, and PCR amplified using Illumina-compatible barcoded primers. PCR product was gel purified on a denaturing polyacrylamide gel. Samples were pooled and sequenced on the Illumina HiSeq platform. iCLIP