Figure 1.

Creation of the cTag-PABP mouse to restrict PAPERCLIP profiling to specific cell populations in intact tissues. (A) Schematics illustrating the strategy for selective PAPERCLIP profiling by expressing “conditionally-tagged” (cTag)-PABP (PABP-GFP) in the targeted cell population (“Tagged” PAPERCLIP, right) and comparing it to the conventional PAPERCLIP (left). (B) Schematics illustrating the targeting strategy for generating the knock-in cTag-PABP mouse (right) and the corresponding Pabpc1 allele nomenclature (left). FNF: Flox-Neo-Flox selection cassette. Black arrowheads denote loxP sites. pA: a synthetic poly(A) site. (C) Genotyping PCR experiments demonstrating successful Cre-mediated excision. Lanes: wildtype (left); Pabpc1^fl (middle); CMV-Cre;Pabpc1^cTag (right). Rapsn serves as a loading control. (D) Fluorescent immunoblots showing Cre-dependent expression of PABP-GFP. Asterisk denotes a non-specific band that was also observed in non-transgenic wildtype mice. (E) Autoradiogram from a cTag-PAPERCLIP experiment. The red arrow denotes the PABP-GFP-RNA complex. The red box shows the area of the nitrocellulose membrane used for RNA extraction and subsequent sequencing library construction. (F) Left, Schematic illustrating the experimental design to compare conventional PAPERCLIP to GFP-PAPERCLIP. Right, a scatter plot showing the correlation of read counts between conventional PAPERCLIP and GFP-PAPERCLIP. Each dot represents a poly(A) site. R², the coefficient of determination.