GSM7927463: IgG_rep2; Homo sapiens; OTHER
Cells were UV-crosslinked and lysed in RIPA buffer. The lysates were sonicated for 10 cycles (30sec on/off) (Diagenode Bioruptor). 500 μg precleared cell protein lysate was treated with 1 U RNase I (Ambion AM2294) for 5 min at 37°C. After addition of 4 μl of RNAsin Plus ribonuclease inhibitor (Promega N2611), the samples were spun down, transferred to a new tube and used for immunoprecipitation (IP) with the antibody coupled beads. The IP eluates were purified after SDS-PAGE electrophoresis and transfer to nitrocellulose membrane, followed by protease digestion and library preparation steps. Libraries were constructed according to Van Nostrand et. al, 2016. The final libraries were prepared following 14 PCR cycles for MDH2 IP and IgG IP and 9 PCR cycles for SMI. The libraries were gel purified, replicates were pooled and analyzed using Agilent Bioanalyzer High-Sensitivity DNA kit and library concentrations were estimated with Qubit.