RNA binding profile of MED23 in 293T cells

To delineate the repertoire of RNA binding of MED23, we performed a denatured CLIP-seq experiment coupled with high-salt wash in MED23-HA 293T cells and wildtype 293T cells as a control. Overall design: The wildtype and MED23-HA cells were crosslinked with UVC (254 nm, 600 mJ/cm2) and then harvested for denaturation by 1% SDS. The denatured cell lysates were diluted by six fold to capture MED23-RNA complex with HA antibody-conjugated beads. The beads were washed with high-stringent buffer and treated with RNase T1 to partially digest RNAs. The RNA fragments were subjected to small RNA library preparations.