Design of RNA/DNA Hybrid IP Method in HeLa Cells
(A) RNA/DNA hybrid IP workflow in HeLa cells.
(B) RNA/DNA hybrid slot blot with S9.6 antibody.
(C) Top: silver stain of RNA/DNA hybrid IP. No antibody and isotype-matched IgG2a antibody were used as controls. Bottom: western blot of RNA/DNA hybrid IP using indicated antibodies. Arrows indicate hypophosphorylated (IIa) and hyperphosphorylated (IIo) forms of Pol II. Triple amounts of input and IP samples were loaded for SETX.
(D) Top: silver stain of RNA/DNA hybrid IP following benzonase treatment. Bottom: western blot of RNA/DNA hybrid IP, probed with Top1 and H3 antibodies.
(E) Silver stain of RNA/DNA hybrid IP in the presence of RNA/DNA hybrid competitor.
(F) Western blot for Top1 of RNA/DNA hybrid IP with indicated synthetic competitors.
(C–E) ∗, indicates the heavy chain of S9.6 and IgG2a antibodies. ∗∗, indicates BSA, used to block protein A dynabeads.
See also Figure S1.