Am9939

Tissue samples were ground in liquid nitrogen using mortar and pestle, followed by homogenization in Ribozol (N580-CA, Amresco) using a polytron homogenizer. Pelleted cultured cells were re-suspended in Ribozol. RNA was isolated by adding chloroform (C2432, Sigma) to the tissue/cell-Ribozol suspension following the manufacturer’s directions. The RNA was resuspended in 20 µL nuclease free water (AM9939, Ambion). RNA quality and quantity was assessed using a QIAxcel Advanced System (Qiagen) and QIAxcel RNA QC Kit v2.0 (Qiagen). From 1 µg of RNA as a template first-strand cDNA was synthesized using qScript cDNA supermix (CA101414-104, Quanta Biosciences). The reverse transcriptase reaction sequence consisted of incubation at 25 °C for 5 min, followed by incubation at 42 °C for 30 min and reverse transcriptase enzyme inactivation by incubation at 85 °C for 5 min. Resulting cDNA samples were stored at −20 °C until further analysis.

Plasmids were prepared by Qiagen plasmid midiprep (#12145) and eluted in nuclease-free water (Ambion Life Technologies AM9939) at a concentration of 500-600 ng/μl. Embryo injection was performed either at the University of Alberta or via GenetiVision Corporation following standard procedures (Fish et al., 2007 (link)). 300-500 embryos per constructs were injected into y¹w*P(nos-PhiC31.NLS;)X;P(carryP)attP40(II) or y¹w*P(nos-PhiC31/int.NLS)X;P(carryP)attP2(III) (for CRISPR/Cas9 constructs) or Bloomington #25709, #25710 (for gRNA constructs). Surviving adults were backcrossed to w¹¹¹⁸ and screened for transformants.