Zymopure plasmid miniprep kit

To clone the Tol2-based transgenesis plasmids, we first amplified the 4,165 bp region upstream of the transcription start site of the betta actb gene using Q5 polymerase (M0491S, New England Biolabs) from an ornamental betta. We then replaced the ubiquitin promoter in the pDestTol2pA2_ubi:EGFP plasmid (27323, Addgene; Mosimann et al., 2011 (link)) with the amplified actb promoter region using bacterial in vivo assembly (García-Nafría et al., 2016 (link)). We then purified plasmids for injection using the ZymoPURE plasmid miniprep kit (D4210, Zymo Research). We fully sequenced all plasmids prior to injection to confirm intended sequences. To create the bicistronic plasmid, we first linearized actb:EGFP and then amplified a 1.3 kb region from pBH mcs (a gift from Michael Nonet) containing the 268 bp of the cmlc2 promoter region, mCherry, and the polyadenylation and large T antigen signals. We then carried out all subsequent steps as described above.

The CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to obtain intermediate constructs by direct PCR and restriction/ligation cloning of required genetic elements. The pRSET-EmGFP plasmid (Thermo Fisher Scientific) was used as an expression vector containing the GFP gene. High-fidelity restriction endonucleases BamHI-HF, BglII-HF, EcoRI-HF, NcoI-HF, and PstI-HF; T4-DNA-ligase; T4-polynucleotide-kinase; and shrimp alkaline phosphatase (rSAP) were purchased from New England Biolabs (Ipswich, MA, USA). The oligonucleotides were synthesised by the solid-phase method and purified by preparative polyacrylamide gel electrophoresis (PAGE) by Syntol LLC (Russia). Q5^® High-Fidelity DNA Polymerase (New England Biolabs) was used for all polymerase chain reaction (PCR). Ultrafree-DA Centrifugal Filter Units (Merck, Kenilworth, NJ, USA) were used for DNA extraction from agarose gel. The ZymoPURE™ Plasmid Miniprep Kit (Zymo Research, Irvine, CA, USA) was used for plasmid DNA purification. The authenticity of the plasmids was confirmed by Sanger sequencing performed by Eurogen CJSC (Russia). E. coli strain NiCo21(DE3) (#C2529H, New England Biolabs, Ipswich, MA, USA) was used for cloning and expression experiments according to the manufacturer’s protocol.

The pcDNA3 GFP LIC vector (a gift from Scott Gradia; Addgene plasmid ##30127) is an empty LIC vector derived from pcDNA3.1(+) which adds a C-terminal GFP gene to the protein of interest. For vector preparation, the pcDNA3 GFP LIC vector was linearized with SspI (New England Biolabs) for 1 hr at 37°C and then purified (DNA Clean-up and Concentration, Zymo Research). The linearized vector was treated with T4-DNA Polymerase (New England Biolabs) and dGTP as per protocol (https://qb3.berkeley.edu/facility/qb3-macrolab/projects/lic-cloning-protocol/) and incubated at 22°C for 40 min. The enzyme was then heat-denatured at 75°C for 20 min. For insert preparation, purified products from the assembly PCR were similarly treated with T4 DNA Polymerase and dCTP per protocol. The annealing reaction was performed by combining 3 µL of treated vector, and 3 µL of treated PCR fragment in 20 µL total volume for 30 min at room temperature. Six µL of the annealing reaction were transformed into One Shot Top10 Chemically Competent cells per manufacturer’s instructions (Thermo Fisher Scientific). Single colonies were prepped (ZymoPURE Plasmid Miniprep Kit, Zymo Research) and proper insertion was confirmed by Sanger sequencing (GeneWiz, South Plainfield, NJ).

The A/B standard sequences were synthesized within pMK vectors by a commercial vendor (ThermoFisher, GeneArt). Following receipt, plasmid sequences were independently confirmed by Sanger sequencing. The plasmids containing the A/B standard sequences were then resuspended in 50 μl nuclease free water and transformed in E. coli as per manufacturer’s protocol (α-Select Competent Cells, Bioline, Australia). The transformed cells were grown overnight (37 °C) in LB agar plate containing Kanamycin (100 μg/ml), after which colonies were selected and further cultured overnight (37 °C; 200 rpm) in 3 ml LB broth also containing Kanamycin (100 μg/ml).
The plasmids with the A/B standards were then extracted and purified using the ZymoPURE Plasmid Miniprep Kit (Zymo Research), according to the manufacturer’s protocol. Purified plasmids were linearized by overnight digestion (37 °C) with EcoRI-HF (NEB) and the products were then visualized on 1% agarose gel. The linear A/B standards were finally treated with Proteinase K and further purified with the Zymo ChIP DCC-25 purification kit (Zymo Research). The final A/B standards were quantified using Qubit dsDNA HS Assay on Qubit 2.0 Fluorometer (Life Technologies) and verified on the Agilent TapeStation with the High Sensitivity DNA Screen Tape Analysis (Agilent Technologies).

The PRPF40A N-terminal region (isoform 2, residues 1-221) was PCR-amplified from RPE1 cDNA (ATCC, Cat# CRL-4000) cDNA. The sequence was amplified in two fragments and both fragments were combined by overhang. All PCRs were carried out using Q5 Polymerase (NEB) according to manufacturer’s protocol. The resulting PRPF40A N-terminal region was cloned into a GFP-expressing pcDNA5 vector (see pcDNA5 GFP vector in ref. ^{28 (link)}) using Q5 Site-Directed Mutagenesis Kit (NEB). Nter-MUT, containing the PPVP/PALPP motifs changed to AAAA/AAAAA, was obtained by mutating the N-ter-WW12-containing plasmid with Q5 site-directed mutagenesis. Plasmids were transformed into DH5alpha chemically competent cells. After culturing, DNA isolation was performed with either ZymoPURE Plasmid Miniprep Kit (Zymo Research) or QIAGEN Plasmid Plus Midi Kit (Qiagen). The sequence was confirmed by Sanger Sequencing (GATC Eurofins). All used oligonucleotides are shown in Supplementary Table 6.

To clone the Tol2-based transgenesis plasmids, we first amplified the 4,165-bp region upstream of the transcription start site of the betta actb gene using Q5 polymerase (M0491S, New England Biolabs) from an ornamental betta. We then replaced the ubiquitin promoter in the pDestTol2pA2_ubi:EGFP plasmid [Addgene plasmid #27323 (Mosimann et al., 2011 (link))]; with the amplified actb promoter region using bacterial in vivo assembly (García-Nafría et al., 2016 (link)). We then purified plasmids for injection using the ZymoPURE plasmid miniprep kit (D4210, Zymo Research). We fully sequenced all plasmids prior to injection to confirm intended sequences. To create the bicistronic plasmid, we first linearized actb:EGFP and then amplified a 1.3-kb region from pBH-mcs (a gift from Michael Nonet) containing the 268-bp of the zebrafish cmlc2 (myl7) promoter region, mCherry, and the polyadenylation and large T antigen signals. We then carried out all subsequent steps as described above.

DNA was manipulated according to the standard protocols with some modifications according to the instructions of reagent manufacturers. Restriction enzymes, T4 DNA ligase, Phusion DNA polymerase, and DNase I were purchased from New England Biolabs. DNA purification was carried out with DNA Clean & Concentrator Kit (Zymo Research). To purify DNA from excised agarose gel, Zymoclean Gel DNA Recovery Kit (Zymo Research) was used. Plasmid DNA isolation was carried out with ZymoPURE Plasmid Miniprep Kit (Zymo Research). Transformation of E. coli XL1-Blue chemically competent cells was carried out by an optimized heat shock method^{52 (link)}.