Whole-mount in situ hybridization protocol

High quality total RNA was extracted and purified as previously described (Kenny et al. 2016 (link)). cDNA was generated by using SMARTer polymerase chain reaction (PCR) cDNA Synthesis kit (Clontech). Gene-specific primers were designed for each gene of interest (supplementary S5, Supplementary Material online). PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). Linearized template DNA (amplified from plasmid with T7/SP6 primers) was used to synthesize RNA probes with either T7 or SP6 RNA polymerase (Life Technologies), and DIG-labeling mix (Roche, Indianapolis, IN, USA). WISH protocol was performed, as previously described (Perry et al. 2015 (link)).

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Truchado-García M., Perry K.J., Cavodeassi F., Kenny N.J., Henry J.Q, & Grande C. (2022). A Small Change With a Twist Ending: A Single Residue in EGF-CFC Drives Bilaterian Asymmetry. Molecular Biology and Evolution, 40(2), msac270.

Publication 2022

SMARTer polymerase chain reaction pGEM-T Easy vector T7 or SP6 RNA polymerase DIG-labeling mix

Cdna Gene Pgem Plasmid Polymerase chain reaction Primers Promega Sp6 rna polymerase Synthesis Vector

Corresponding Organization : University of California, Berkeley

Other organizations : University of Illinois Urbana-Champaign, St George's, University of London, Natural History Museum, University of Otago, Marine Biological Laboratory

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Variable analysis

independent variables

Gene-specific primers designed for each gene of interest

dependent variables

Gene expression patterns as measured by WISH (Whole-mount in situ hybridization) protocol

control variables

High quality total RNA was extracted and purified as previously described (Kenny et al. 2016)
CDNA was generated by using SMARTer polymerase chain reaction (PCR) cDNA Synthesis kit (Clontech)
PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA)
Linearized template DNA (amplified from plasmid with T7/SP6 primers) was used to synthesize RNA probes with either T7 or SP6 RNA polymerase (Life Technologies), and DIG-labeling mix (Roche, Indianapolis, IN, USA)
WISH protocol was performed, as previously described (Perry et al. 2015)

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