FLAG-tagged FBF-1 and FBF-2 transgenes used for iCLIP in intact animals. (A) Transgenes encoding FLAG-tagged FBF-1 and FBF-2. Depicted gene regions possess wild-type fbf-1 or fbf-2 sequences with the addition of N-terminal triple FLAG tag. These constructs were incorporated into the C. elegans genome as single copies (see Materials and Methods). (B) Approximate abundance of FLAG-tagged FBF-1 and FBF-2, assayed by Western blot stained with anti-FLAG antibody. Lysates were prepared from UV crosslinked transgenic animals [FBF-1: fbf-1(0) 3xflag::fbf-1 and FBF-2: fbf-2(0) 3xflag::fbf-2] or from control animals treated identically (wild-type N2). (C) Depletion of FLAG-tagged FBF-1 and FBF-2 after IP, assayed by Western blot from transgenic animals as in B. (D) iCLIP workflow begins with live animals and ends with high-throughput sequencing to elucidate the genome-wide targets of FBF-1 and FBF-2.
