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Supplementary experimental procedures
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Preparation of RNA for 5’ RACE (Rapid Amplification of cDNA Ends) was performed using the
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GeneRacer kit (Life Technologies, Grand Island, NY). Reverse transcription for RACE was
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performed using the SuperScript III RT kit and RACE reactions were performed using Platinum Taq
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DNA polymerase according to the manufacturer’s instructions (Life Technologies, Grand Island,
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NY) with gene-specific primers (Table S1). PCR products of interest were separated by gel
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electrophoresis, excised from the gel, and purified using the QIAEX II gel extraction kit (Qiagen,
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Valencia, CA). The purified PCR products were cloned using the TOPO TA Cloning Kit for
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Sequencing and transformed into TOP-10 chemically competent E. coli cells (Life Technologies,
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Grand Island, NY). The resulting transformants were prepared for sequencing with the QIAprep spin
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miniprep kit (Qiagen, Valencia, CA) and screened for the correct insert by plasmid digestion with
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EcoRI (Fermentas, Gen Burnie, MD). Plasmid inserts were sequenced using the M13F and -R
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primers (Life Technologies, Grand Island, NY).
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RNA was extracted from light organs using the RNeasy Fibrous Tissue Kit (Qiagen,
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Valencia, CA) after homogenizing the organs in a TissueLyser LT (Qiagen, Valencia, CA). Three to
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four biological replicates were used per experiment. The extracts were treated with the Ambion
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TURBO DNA-free kit (Life Technologies, Grand Island, NY) to remove any contaminating genomic
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DNA. The RNA extracts were then quantified using a Qubit 2.0 Fluorometer (Life Technologies,
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Grand Island, NY) and 5 μL of each preparation were separated on a 1% agarose gel to ensure the
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integrity of the RNA. If not used immediately, the RNA extracts were aliquoted and stored at -80°C.
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cDNA synthesis was performed with SMART MMLV Reverse Transcriptase (Clontech, Mountain
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View, CA) according to the manufacturer’s instructions, and then each cDNA synthesis reaction was
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diluted to a concentration of 2.08 ng/μL using nuclease-free water and stored at 4°C.
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For each qRT-PCR experiment, wells without a template and with cDNA reactions run with
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no reverse transcriptase as a template were run as negative controls to ensure the absence of
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chromosomal DNA in the reaction wells. The efficiencies of all qRT-PCR primer sets were between
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95 and 105%. Data were analyzed using the ΔΔCq method (Pfaffl 2001). qRT-PCR was performed
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on light organ cDNA using iQSYBR Green Supermix or SsoAdvanced SYBR Green Supermix
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(BioRad, Hercules, CA) in a CFX Connect Real-Time System (BioRad, Hercules, CA).
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Amplification was performed under the following conditions: 95°C for 5 min, followed by 45 cycles
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of 95°C for 15 sec, 60°C for 15 sec, and 72°C for 15 sec. Each reaction was performed in duplicate
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and contained 0.2 μM primers and 10.4 ng of cDNA. To determine whether each PCR reaction
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resulted in a single amplicon, the presence for one optimal dissociation temperature for each PCR
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reaction was assayed by incrementally increasing the temperature every 10 sec from 60 to 89.5°C.
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Each reaction in this study had a single dissociation peak. Standard curves were created using a 10-
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fold dilution of the PCR product with each primer set.
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For Western-blot analyses, concentrations of the protein samples were determined using a
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Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY). The proteins were separated on
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12.5% SDS-PAGE gel with 25 μg of protein per lane and then transferred onto a PVDF membrane
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with a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad, Hercules, CA) per the manufacturer’s
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sinstructions. The membrane was blocked overnight at room temperature (RT) as previously
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described (Troll et al 2010). The antibody was diluted 1:1000 in blocking solution and incubated
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with the membrane for 3 h at RT. The blot was then exposed to secondary antibody, washed, and
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developed as previously described (Troll et al 2010).
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In ICC experiments, the organs were incubated with a 1:500 dilution of the EsGal1 antibody
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in blocking solution of 1% goat serum, 1% TritonX100, and 0.5% bovine serum albumin in marine
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PBS (50 mM sodium phosphate, 0.45 M sodium chloride, pH 7.4) for 7 to 14 d at 4°C, and then
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rinsed 4 x 1 h in 1% Triton X-100 in marine PBS, and incubated overnight in blocking solution at
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4°C. Samples were then incubated with a 1:50 dilution of a fluorescein-conjugated goat anti-rabbit
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secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) in blocking solution
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in the dark at 4°C overnight.
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For mucus secretion assays, juvenile squid were fixed in Bouin’s solution for 3 h at room
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temperature and then washed 4 x 1 h in mPBS at 4°C. The samples were then treated as for the
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above ICC experiments, except for the use of wheat germ agglutinin as a counterstain to visualize
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host mucus.
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