RNA-protein interactions were characterized using the iCLIP protcol (cross-linking and immunoprecipitation). RNA was UV-crosslinked to protein, isolated by immunoprecipitation using msl-2 antibody, and characterized by deep sequencing analysis.

Source was cell extract of clone8 cell line; bait produced from endogenous gene; prey produced from endogenous gene.

Though msl-2 bound to 5,206 sites, roX1 and roX2 sites scored considerably higher, representing over half of all observed cross-linking events.

RNA bait was tagged with MS2 stem loops, and immobilized on amylose beads using a fusion of MS2-binding protein and maltose binding protein (MBP).

Interaction in vitro; bait produced by in vitro transcription; prey produced as a recombinant fusion protein in baculovirus-infected Sf21 cell system.

Interaction in vitro; bait produced by in vitro transcription; prey produced as a recombinant fusion protein in baculovirus-infected Sf21 cell system.

In the presence of ATP, mle and msl-2 form higher order complexes with roX2.

Interaction in vitro; bait produced and labeled by in vitro transcription; prey produced as a recombinant fusion protein in bacterial system.

positive control

Chromatin cross-linked RNA was incubated with biotin-labeled lncRNA:roX2 probes. msl-2-lncRNA:roX2 complexes were pulled down with streptavadin.

Source was cell extract of S2 cell line; bait and prey produced from endogenous gene.