Intestinal Single-cell Gene Response to Toxins

To investigate single cell intestinal gene responses to toxic treatments, adult A. suum were treated using a protocol employed in other contexts [36 (link)]. Briefly, the method involved injection of treatments through the body wall of adult A. suum, midway down the length, and into the pseudocoelomic space using a 28-gauge needle. Accuracy of injections was monitored under a dissecting microscope. The volumes of injected solutions were adjusted to 1% of weight of recipient worms, such that the diluent (DMSO) did not exceed 1% of the body weight and final concentrations of toxic treatments were 500 µM CID 1067700 (SML054) or Leflunomide (L5025), (Sigma-Aldrich, St. Louis, MO), based on previous applications with these nematode intestinal toxins/toxicants (NITs [6 (link), 7 (link)]). “CID” stands for the “compound identification” number used on PubChem [37 (link)], as described in the original study identifying it [38 (link)]. Control worms received DMSO only. Two adult female worms injected for each treatment were next incubated for 2 h in PBS (37^oC) under ambient air conditions. Intestinal samples were then obtained by dissection and processed for scRNA-seq as described above for untreated worms and shipped to Washington University for single cell nuclei preparations.