Bsphi

All zebrafish work followed protocols approved by Columbia University Irving Medical Center’s Institutional Animal Care and Use Committee. We obtained cpsf6^sa9322 from the Zebrafish International Resource Center (ZIRC) (72 (link)). This allele has a point mutation (C>T) creating a premature stop codon in exon 4. To genotype adult fish, we extracted genomic DNA from the fin; for paraformaldehyde (PFA)–fixed larvae, we used whole larva for genotyping. Fins or larvae were dissolved in 50 or 20 μl of 50 mM NaOH, respectively, incubated for 20 min at 95°C neutralized with tris-HCl (pH 7.5; 1:10). We performed PCR with EconoTaq PLUS GREEN (Lucigen, Middleton, WI, catalog no. 30033-1) according to the manufacturer’s instructions, using between 2 and 5 μl of genomic DNA and the following primers: cpsf6-fw 5′-GCCTTCCCCTCTCCTGACAT-3′ and cpsf6-rv 5′-ATCTCTCTGTGGCCCTCACC-3′. Two microliters of the 666-bp PCR product was digested with 0.3 μl of Bsph I (New England Biolabs, catalog no. R0517). The C>T mutation creates a Bsph I restriction site; the PCR product from wild-type zebrafish will result in one DNA band (666 bp), cpsf6^+/− will result in three bands (666, 466, and 200 bp), and cpsf6^−/− will result in two bands (466 and 200 bp).

DNA strands were purchased from Sangon (Shanghai, China), and the sequences are listed in the supporting information. Tris(hydroxymethyl)aminomethane hydrochloride (Tris) was purchased from Bio-Rad (Singapore); MgCl₂ were from Quality Reagent Chemical (Singapore); EDTA were from USB biochemical (USA); agarose was purchased from Bio-Rad (Singapore); 100bp DNA ladder was from Thermo Fisher (Singapore); Endonuclease MspI and BspHI were purchased from New England Biolabs (Singapore). Ultrapure water with 18.2 MΩ·cm was used in all experiments.