Cloning and Tagging Killifish Hormone Genes

Killifish cDNAs were cloned from brain tissues from male and female killifish by homogenization in TRIreagent (Sigma #T9424) using 3 mm Nirosta disruption beads (PALBOREG FEDERAL #BL6693003000) and a tissue homogenizer (TissueLyser LT, Qiagen #85600). Total RNA was isolated from the lysed tissues using the Direct-zol RNA miniprep kit. (Zymo research #R2052), and the Verso cDNA Synthesis Kit (Thermo scientific #AB1453A) was used to prepare cDNA with random primers according to the manufacturer’s protocol. cDNA for the gh1 and fshb was amplified using custom DNA oligonucleotides (Sigma) and Platinum SuperFi II DNA Polymerase (Invitrogen, #12361010). Primer sequences are available in the Key Resources Table. PCR products were purified (QIAquick PCR purification kit, Qiagen #28104) and sequence-verified. The sequence-verified ORFs were cloned using GIBSON (NEB, #E2611L) into the pLV-EGFP plasmid or our Dox inducible plasmid (#196331), which was modified such that each hormone is tagged with a GFP, separated by the T2A self-cleaving peptide (Liu et al., 2017 (link)). Plasmids and corresponding annotated maps are available via Addgene (#194356, #194883, #196331, # 205595, # 205596, #205597).