A sea bream (Sparus auratus) Pac1a construct was available in house (Cardoso et al., 2007) and was used to infer the activity of the tilapia receptor homologue as they shared over 90% aa sequence identity (Supplementary Figure 2). The complete coding sequence of tilapia vipr2a was amplified from brain cDNA using specific primers and proofreading DNA polymerase (iProof, BioRad, Portugal, Supplementary Table 1). The PCR thermocycle consisted of: 98 ºC for 30 sec, followed by 45 cycles (98 ºC 10 sec, 64 ºC for 30 sec, 72 ºC for 2 min) with a final extension of 72 ºC for 10 min. The PCR product was cloned into the pcDNA3.1 vector (Directional TOPO Expression Kit, Invitrogen, USA) and used to transform TOP10 competent bacteria. Positive bacterial colonies were screened by PCR using receptor specific primers. Plasmid DNA was isolated from those giving a PCR product of the expected size using the standard alkaline lysis method and was sequenced to confirm identity. Human RAMP1 (conceded by Dr Vanessa Schein, UFRGS, Brazil) was amplified with proofreading DNA polymerase (iProof, BioRad, Portugal) using specific primers (Supplementary Table 1) and cloned into pcDNA3.1.
Iproof
Manufactured by Bio-Rad
Sourced in Portugal
The IProof is a thermal cycler instrument designed for polymerase chain reaction (PCR) amplification of DNA samples. It provides precise temperature control and reliable performance for a variety of PCR applications.
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Lab products found in correlation
End repair kit, by Illumina (2 mentions)
Paired end adapters, by Qiagen (2 mentions)
Pe 102 1003, by Qiagen (2 mentions)
Ultrapure ligase l603 hc l, by Qiagen (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Dneasy blood and tissue kit, by Covaris (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Snapgene software, by GSL Biotech (1 mentions)
Snap tag t7 2vector, by New England Biolabs (1 mentions)
La taq, by Takara Bio (1 mentions)
Hotstartaq plus, by Qiagen (1 mentions)
Genomewalker universal kit, by Takara Bio (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Lr clonase 2, by Thermo Fisher Scientific (1 mentions)
Pdonr221, by Thermo Fisher Scientific (1 mentions)
Bp clonase 2, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Directional topo expression kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using iproof
1
Cloning and Characterization of Tilapia Vipr2a
2
Formin Actin Assembly Domains Expressed in Bacteria
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Constructs containing different components of the formin actin assembly domains (FH1 and FH2) were prepared for bacterial expression. The preparation of Cdc12(FH1FH2) and Cdc12(FH1) constructs has been described (Neidt et al., 2009 (link)). The FOR1 domain constructs were designed based on sequence analysis of the Chlamydomonas genome and Expressed Sequence Tag analysis by Susan Dutcher (Washington University, St. Louis, MO) and were optimized for bacterial expression and custom synthesized (DNA 2.0, Newark, CA). Constructs were designed by SnapGene software (from GSL Biotech; available at snapgene.com). All constructs were prepared by standard cloning procedures, consisting of PCR amplification (iProof, Bio-Rad Laboratories) from the commercially prepared DNA. Restriction enzyme cleavage sites and 6× His sequences were included in the reverse primers. PCR products were cloned using restriction enzymes into pET21a (EMD Biosciences) for expression. All amplified sequences were confirmed by sequencing.
3
Illumina Sequencing Library Preparation
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DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit and sheared using a Covaris E220 (Woburn, MA) (duty cycle 10, intensity 5, cycles/burst 200 and time 180 s) to ~400 base pair (bp). Sheared genomic DNA was gel purified and used as input for Illumina sequencing library prep, with end-repair using the NEBNext end-repair kit (E6050L), and A-tailing with Taq polymerase. Ligation to Illumina paired-end adapters (PE‐102-1003) was done using Ultrapure ligase (L603-HC-L) from Enzymatics, and amplification with iProof (Bio-Rad). Agencourt Ampure XP beads were used for clean-up and size selection at each step. The resulting DNA library was sequenced on a single lane of an Illumina HiSeq2000 at the Stanford Center for Genomics and Personalized Medicine.
4
Production and Cloning of VopL/F Constructs
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VopL/F constructs were prepared by infusion (Takara Bio Inc.) after PCR amplification (iProof; Bio-Rad Laboratories) from plasmid DNA for VopL and from codon-optimized plasmid DNA for VopF (provided by E. Kerkhoff, University of Regensburg, Regensburg, Germany), with a 6xHis tag included in the reverse primer. PCR products were cloned into the SNAP-tag-T7-2vector (New England Biolabs, Inc.) at the XmaI/PacI sites, but the PacI site is not maintained. A flexible linker (GGSGGS) was included in the forward primer sequences of the SNAP constructs between the SNAP and the VopL/F DNA sequences.
5
Bimolecular Fluorescence Complementation Analysis
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BiFC was performed as described previously (Zhong et al., 2008 ). MRE11 and TAF1(1278–1919) were amplified and cloned into the entry vector pENTRE (Invitrogen) before subcloning into the split YFP vectors pDH51‐GW‐YFPn and pDH51‐GW‐YFPc. Truncations of TAF1 were made by restriction digestion: the bromodomain‐YFP fusion (TAF1 1755–1919) was constructed using XbaI–NotI digestion followed by ligation with the filler oligonucleotide GGATATG. The bromodomain was removed (TAF 1246–1606) by NsiI–XhoI digestion followed by DNA polymerase fill in (iProof; Bio‐Rad) and ligation. Purified plasmids were used to transform Arabidopsis protoplasts according to the tape‐sandwich method (Wu et al., 2009 ). Fluorescence imaging was performed on a Zeiss (www.zeiss.co.uk ) Axiovert 700 inverted confocal microscope.
6
Cloning and Sequencing of Opsin Genes
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For two opsins (Platynereis dumerilii ciliary opsin (AAV63834.1) and Leptochiton asellus xenopsin) the whole genes were cloned from genomic DNA for subsequent analysis of exon-intron boundaries. Genomic DNA was extracted with the Nucleospin Tissue Kit (Machery-Nagel, Düren, Germany) and tested for fragment lengths larger than 20 kb. As a starting point, gene-specific primers were designed on the basis of the transcript sequences. For genome walking, four libraries were prepared with the Universal Genome Walker Kit (Takara Bio, Kusatsu, Japan) by enzymatic digestion and used for sequence elongation starting from exonic fragments. In parallel, long amplicons bridging smaller introns were also directly amplified from genomic DNA using Lataq (Takara Bio), iProof (Biorad, Hercules, California) and HotStarTaq Plus (Qiagen, Hilden, Germany, RRID:SCR_008539 ) polymerases. Obtained amplicons of up to 8 kb were cloned using a pGem-T easy Vector (Promega) TOPO XL PCR cloning kit (Thermo Fisher Scientific), TopTen chemically competent cells (Thermo Fisher Scientific) and Sanger sequenced. Obtained sequences were used to design further primers for ongoing sequence elongation. Read assembly was performed with CLC Main Workstation (RRID:SCR_000354 ) 7.1.
7
DNA Extraction, Shearing, and Library Prep
8
gRNA Library Generation and Validation
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The gRNA oligonucleotide library was synthesized by Agilent and resuspended at 1 ng/μL in water. All library amplifications were done using iProof (Bio-Rad), using 2.5 ng of the oligonucleotide pool as template per 50 μL reaction. Sublibraries were amplified using primers P40/41 for library 1 and P42/43 for library 2, and subsequently amplified with primers P44/45 for cloning. Amplified libraries were Gibson assembled into gel-extracted (Zymo) BsaI-digested pU6_Library_DHFR, dialyzed against water, and electroporated into E. cloni electrocompetent cells (Lucigen) according to manufacturer’s protocol. Coverage was assessed by dilution plating in comparison to a no-insert negative control. Libraries were maxiprepped (Zymo), and retransformed into chemically competent NEB 5-alpha (NEB) to improve yields for transfection. Both E. cloni and NEB 5-alpha libraries were sequenced to ensure diversity. Libraries were linearized with AseI, dialyzed 1 h against water, and divided into 50 μg aliquots. Guide RNAs against mNeonGreen were assembled separately by annealing primer pairs P46/47, P48/49, P50/51, P52/53, or P54/55 and Gibson assembling into gel-extracted, BsaI-digested pU6_Library_DHFR. Constructs were verified by sequencing with P19, and spiked into library aliquots at equimolar concentrations.
9
Lentiviral Inducible Gene Overexpression
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To generate a lentiviral transfer plasmid for inducible gain-of-function experiments, we used high-fidelity PCR (iProof, BioRad) to amplify full-length mouse COUP-TFII cDNA, flanked with attB1 and attB2 sites on 5' and 3' ends respectively, from a cDNA library from adult mouse kidney. The resulting PCR band was purified from a 0.8% agarose gel using QIAquick Gel Extraction kit (Qiagen). The purified PCR product was then cloned into pDONR221 (Thermo Fisher Scientific) using BP Clonase II (Invitrogen) according to manufacturer's instructions. We then shuttled the COUP-TFII cDNA into the destination vector pInducer20 (a gift from Stephen Elledge, Addgene #44010) using LR Clonase II (Invitrogen). All cloning steps were verified using Sanger sequencing (Genewiz, Inc.). C3H10T1/2 cells were infected with lentivirus in the presence of 10 µg/ml polybrene (Sigma). Infected cells were selected with puromycin (Sigma).
10
Genomic DNA Extraction and Amplification
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Harvested cells were treated with proteinase K (50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl 2, 0.1 mg/mL gelatin, 0.45% (v/v) NP40, 0.45% (v/v) Tween-20, 0.5 mg/ml proteinase K (Roche)) for 90 min at 55 C. After heat-inactivation of the enzyme (10 min at 95 C) PCR was performed with iProof (Biorad) according to manufacturer's instructions. Primers for amplification and sequencing of genomic DNA were as follows:
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