GSM9823024: CCT3-2; Mus musculus; RNA-Seq
Cell pellets were resuspended in eCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1 mM PMSF, protease inhibitor cocktail and RNase inhibitor (Superase-in). Lysates were sonicated on ice and treated with RNase I to fragment RNA. Immunoprecipitation was performed using anti-PABPC1 antibody and anti-CCT3 antibody conjugated to protein G magnetic beads overnight at 4°C eCLIP libraries were constructed following the standard enhanced crosslinking immunoprecipitation protocol. Briefly, RNA fragments bound to beads were dephosphorylated, ligated to 3' RNA adapters, reverse transcribed with barcoded primers, and PCR amplified. Input control libraries were prepared in parallel from the same cell lysates without immunoprecipitation