GSM9157451: YBX1 eCLIP, 19-28z T cells, IP2; Homo sapiens; OTHER
Cell pellets were lysed using 1mL of eCLIP lysis mix with a modified composition of 6µL of Proteinase Inhibitor Cocktail and 20µL of Murine RNase Inhibitor. Samples were then subjected to one round of sonication for 5 minutes with 30 second ON/OFF at 75% amplitude. Pre-validated antibodies against YBX1 (Abcam cat. Ab76149) were then pre-coupled to Anti-Rabbit IgG Dynabeads (ThermoFisher), added to lysate, and incubated overnight at 4°C. Prior to immunoprecipitation, 2% of the sample was taken as the paired input sample, with the remainder magnetically separated and washed with eCLIP high stringency wash buffers. IP and input samples were cut from the membrane at the relative band size to 75kDa above. RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed.