Dig labeling kit

To generate a template for zrsr2 riboprobe synthesis for WISH, we performed RT-PCR using RNA from 5 dpf WT embryos and primers from the region shared by both isoforms (Supplementary Table S1). The RT-PCR product was cloned into a pCR4-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequence-verified by Sanger sequencing. The plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD, USA), digested with SpeI, and labeled with a DIG labeling kit (Sigma-Aldrich, St. Louis, MO, USA) using T7 polymerase. Antisense riboprobes for all other WISH markers used in this study were also generated using a DIG labeling kit (Sigma-Aldrich, St. Louis, MO, USA). Zrsr2^hg129/+ fish were in-crossed, and embryos were collected at various developmental stages. Collected embryos were euthanized and fixed overnight in 4% PFA at 4 °C. WISH was performed as described previously [44 (link)]. For o-dianisidine staining, euthanized embryos were incubated in o-dianisidine (MP Biomedicals, Costa Mesa, CA, USA) solution for 15 min in the dark [45 (link)], followed by fixation in 4% PFA prior to imaging. Embryos used for WISH and o-dianisidine staining were genotyped via fluorescent PCR, as described previously [41 (link)], using REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) for product amplification.

Template DNAs for RNA probes were synthesized by PCR amplification of a part of genes cloned in the previous section. Primers used can be found in Table S2. After gel purification, the DNA template concentration should be at least 100 ng/µl. The transcription using T7 RNA polymerase was carried with the DIG labeling Kit (Sigma-Aldrich, Cat No. 11175025910). In vitro transcription mixture was prepared by adding 2 µl of acetylated BSA, 2 µl of transcription buffer, 2 µl of 10% Triton X-100, 2 µl of 10XNTP labeling mixture and 1 µl of RNase inhibitor with a final volume of 9 µl. To this mixture, 9 µl of purified PCR product and 2 µl of T7 RNA polymerase were added. The reaction was incubated at 37°C for 3 h and then DNA was removed from the mixture by adding 5 µl of DNase buffer, 22 µl of RNase Free water and 2 µl of RNase-free DNase. The mixture was incubated for 15 min. at 37°C. Finally, the probe was let to precipitate overnight at −80°C by adding 6 µl of 4 M LiCl, 1 µl of 0.5 M EDTA and 180 µl of 100% ethanol. The next day, RNA was precipitated by centrifugation at 13,000 rpm for 30 min. at 4°C. RNA pellet was washed with ethanol, dried, and dissolved in RNase-free water.