PAT-Seq of intestine and muscles tissues. (A) PAT-Seq uses Gateway-compatible (GW) entry vectors expressing the PolyA-Pull cassette in each tissue using tissue-specific (TS) promoters. (1) PolyA-Pull expressed in the intestine (ges-1 promoter), pharynx (myo-2), and body muscle (myo-3). (2) Expression of PolyA-Pull produces a 3 × FLAG-tag (light blue) fused to PAB-1 (blue), which specifically binds to the poly(A) tails of mRNAs (TS mRNAs). The complex is immunoprecipitated using α-FLAG beads. (3) Tissue-specific cDNA libraries are sequenced and mapped onto the WS190 gene model. (B) Detection of stable integration of the PolyA-Pull cassette. Left panel: Using PCR we detected genomic integration of the common portion of the PolyA-Pull cassette (2.6 kb band, red asterisk) in each tissue. The negative control, myo-2Δpab-1, was also integrated. Right panel: Western blotting using α-FLAG antibodies detected the in-frame PolyA-Pull fusion protein in lysates from transgenic worms expressing it in the pharynx (myo-2::pab-1) but not in lysate from wild type N2 worms. (C) Quantification of the specificity and sensitivity of the pull-down using RT-PCR: Left panel: myo-2 (lane 1) (*), ges-1 (lane 3) (**) and dpy-7 (lane 4) transcripts were detected in total RNA extracted from wild type N2 worms. Middle panel: Using immunoprecipitation, we successfully detected the presence of the muscle-specific gene myo-2 (lane 5) (*) and the exogenous unc-54 3’UTR (lane 6), but not the intestine-specific ges-1 (lane 7) (**) and the hypodermis-specific dpy-7 (lane 8). These transgenic worms expressed PolyA-Pull cassette in the pharynx, but not in our negative control in wild type N2 worms (lanes 9-12). The primers used to detect unc-54 3’UTR also detected 18S rRNAs (lane 2). This band was replaced with two unc-54 3’UTR isoforms (lane 6), suggesting that PolyA-Pull enriched for polyA+ RNAs. Right panel: We are unable to isolate tissue-specific RNA from worms lacking pab-1 (Δpab-1).