The three zebrafish TG2 coding sequences have been cloned in the pET30a(+) bacterial expression vector in order to produce the three TGs2 as His6-tagged recombinant proteins. Zebrafish TGs sequences were PCR-amplified with Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA USA, cat#F530S), using 2 dpf zebrafish embryos cDNA as template and zTGs2-specific primers, designed to insert restriction sites for KpnI and HindIII, respectively, at the 5′ and 3′ of the coding sequences (Table 2 ). The PCR products were gel purified with the GenElute™ Gel Extraction Kit (Sigma-Aldrich, Burlington, MA, USA, cat#NA1010), following the manufacturer’s instructions, and digested with KpnI and HindIII (New England BioLabs, Ipswich, MA, USA, cat#R3142 and cat#R3104) together with the recipient plasmid. The digested vector and DNA fragments were ligated with T4 DNA ligase (Thermo Scientific, cat#EL0011) and cloned into DH5α competent cells. Plasmid minipreps were obtained from overnight culture at 37 °C in 2xTY medium with GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, cat#PLN70), and transformed into BL21(DE3) competent cells.
Genelute gel extraction kit
Manufactured by Merck Group
Sourced in United States, Germany, Canada
The GenElute Gel Extraction Kit is a laboratory product designed to extract and purify DNA fragments from agarose gels. It facilitates the isolation and recovery of DNA samples from electrophoresis gels for further analysis or applications.
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Lab products found in correlation
Genelute plasmid miniprep kit, by Merck Group (6 mentions)
Genelute pcr clean up kit, by Merck Group (5 mentions)
T4 dna ligase, by New England Biolabs (4 mentions)
Clonejet pcr cloning kit, by Thermo Fisher Scientific (4 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (3 mentions)
Topo ta cloning kit for sequencing, by Thermo Fisher Scientific (3 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (2 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)
Abi prism 3130 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Onestep rt pcr enzyme mix, by Qiagen (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Pgem t easy cloning vector, by Promega (2 mentions)
One shot top10 chemically competent e coli cells, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 v5 his topo ta expression vector, by Thermo Fisher Scientific (2 mentions)
Genelute mammalian genomic dna miniprep kit, by Merck Group (2 mentions)
Genelute hp plasmid miniprep kit, by Merck Group (2 mentions)
84 protocols using genelute gel extraction kit
1
Recombinant Zebrafish TG2 Protein Production
2
Characterization of Arabidopsis Mutant Alleles
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We obtained Arabidopsis Columbia-0 seeds from the Nottingham Arabidopsis Stock Center. Single, double and triple mutants between hws-1, cuc1-1D and cuc2-1D were generated by crossing the genotypes as described in [27 ]. The ffo1 mutant (Landsberg background) [28 (link)] was sourced from Elliot Meyerowitz, and the cuc2-1D mutant (Columbia-0 background) [24 (link)] from John Walker. The F1 plants were self-pollinated and homozygous F2 lines were identified using PCR. All lines were grown in a growth room with temperature of 22±2°C, and photoperiod of 22h light/2h darkness supplemented with fluorescent lights at a light intensity of 200 μmol m-2s-1 (Polylox XK 58W G-E 93331). The hws-1 EMS populations were maintained in a greenhouse, temperature: 23±2°C and photoperiod: 16h light/8h darkness.
To identify the nature of the mutation in allele hws-5 (ffo1), genomic DNA from seedlings from the ffo1 mutant was extracted (Qiagen, DNAeasy Plant Mini kit) and used to amplify the genomic region from the HWS gene using the primers At3g61590ForcDNA and At3g61590rev. PCR reactions were performed using Platinum® pfx DNA polymerase (Invitrogen). The amplified band was gel purified using Genelute™ Gel extraction kit (Sigma), and sequenced using primers At3g61590ForcDNA, At3g61590Rev, SSLPHSFor, SSLPHSRev, HS 5’endutrfor and HSmap3rev.
To identify the nature of the mutation in allele hws-5 (ffo1), genomic DNA from seedlings from the ffo1 mutant was extracted (Qiagen, DNAeasy Plant Mini kit) and used to amplify the genomic region from the HWS gene using the primers At3g61590ForcDNA and At3g61590rev. PCR reactions were performed using Platinum® pfx DNA polymerase (Invitrogen). The amplified band was gel purified using Genelute™ Gel extraction kit (Sigma), and sequenced using primers At3g61590ForcDNA, At3g61590Rev, SSLPHSFor, SSLPHSRev, HS 5’endutrfor and HSmap3rev.
3
Zebrafish embryo manipulation by morpholino
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Embryos from wild-type AB strain were obtained by natural spawning and raised in Petri dishes at 28.5°C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). Embryos were treated with phenylthiourea to inhibit pigmentation, and collected at different developmental stages.
The splice-morpholino, atg4daSMO, binding to intron1 and exon2 of atg4da pre-mRNA, and a stdMO were purchased from Gene Tools (S7 Table ). The morpholinos were dissolved in 1x Danieu’s solution and approximately 2–3 ng/embryo were injected into 1-cell stage embryos. The specificity of the splice morpholino was evaluated by RT-PCR using primers listed in S7 Table . The PCR products were visualized on 3% agarose gel and processed for sequencing using the GenElute Gel Extraction Kit (Sigma-Aldrich). The used zebrafish atg4da reference sequences were ENSDART00000152289 (mRNA) and ENSDARP00000126975 (protein).
The splice-morpholino, atg4daSMO, binding to intron1 and exon2 of atg4da pre-mRNA, and a stdMO were purchased from Gene Tools (
4
DNA Sequencing of Purified PCR Products
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The PCR products were separated by electrophoresis in 2% agarose gel and photographed for future densitometric analysis, which was performed with ImageJ [39 ]. Bands of interest were cut out of the gel and purified using a GenElute Gel Extraction Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. After being eluted from the column, 10 µL of eluate was loaded in 2% agarose gel in order to determine the purity and amount of eluate that was going to be used in the sequencing reaction. Only the amplicon corresponding to TV4 needed to be purified, re-amplified one more time with NQO1F/NQO1R and purified again. A sufficient amount of the template was obtained for the sequencing reaction only after this additional procedure.
The sequencing reaction contained 12 µL mixture of the purified PCR product (approximately 50 ng per 100 bps), nuclease-free water and 1 µL of the forward and reverse primer used in PCR, respectively (primer concentration 3.2 pmol/µL), for a total volume of 13 µL. The samples were sequenced at the DNA Sequencing Core Facility of the Rudjer Boskovic Institute.
The sequencing reaction contained 12 µL mixture of the purified PCR product (approximately 50 ng per 100 bps), nuclease-free water and 1 µL of the forward and reverse primer used in PCR, respectively (primer concentration 3.2 pmol/µL), for a total volume of 13 µL. The samples were sequenced at the DNA Sequencing Core Facility of the Rudjer Boskovic Institute.
5
Sequencing of the GNAI3 Gene in Ocular Albinism
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Twenty-two sets of primers (forward and reverse) were designed for sequencing the 5’ flanking region, each of the 9 exons, and the 3’ UTR of the GNAI3 gene in the genomic DNA samples from the 26 patients with reported ocular albinism who did not have a mutation in the OA1 gene (Table 1 ) and from the 6 control individuals. While only one set of primers was used for amplification of the 5’ UTR and exons 1 through 8, 14 sets of primers were required to amplify the long, non-coding exon 9/3’UTR. We tested and optimized each primer by PCR using DNA from a normal individual, and the sizes of the PCR products were verified by electrophoresis on 2% agarose gels. Two different PCR programs (#1 or #2), varying only in the temperature of step 3, allowed optimal amplification of the corresponding fragments:
1. 95°C for 3 min; 2. 95°C for 45 sec; 3. #1, 58°C for 45 sec; #2, 47°C (50°C for set 5) for 45 sec; 4. 72°C for 1 min; 5. Repeat 34 times steps 2–4. 6. 72°C for 5 min. For PCR products with two or more amplified bands, the desired band size was cut from the gel and its DNA was extracted with the GenElute Gel Extraction kit (Sigma Aldrich). The eluted DNA was concentrated and then re-suspended in 12 μl of exotoxin-free water.
1. 95°C for 3 min; 2. 95°C for 45 sec; 3. #1, 58°C for 45 sec; #2, 47°C (50°C for set 5) for 45 sec; 4. 72°C for 1 min; 5. Repeat 34 times steps 2–4. 6. 72°C for 5 min. For PCR products with two or more amplified bands, the desired band size was cut from the gel and its DNA was extracted with the GenElute Gel Extraction kit (Sigma Aldrich). The eluted DNA was concentrated and then re-suspended in 12 μl of exotoxin-free water.
6
Protein Expression from Engineered DNA
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For the template, DNA was isolated from derivatives of pEGFP-N2 containing one, five or nine codons of fHbp fused to GFP (GenElute Gel Extraction Kit, Sigma), linearised by digestion with NotI (New England Biolabs) and then purified using QIAquick (Qiagen). For RNA and DNA templates, 1 μg of nucleic acid was used as a template. The in vitro transcription/translation reaction was performed at 30°C, 37°C or 42°C for one hour using the E. coli S30 Extract system for Linear Templates in vitro Transcription/Translation Kit (Promega). The products were precipitated in acetone, then re-suspended in SDS-PAGE buffer prior to Western blot analysis.
RNA sequences were analysed using the RNAfold web server of the Vienna RNA package (http://rna.tbi.univie.ac.at/egi-bin/RNAfold.cgi ). For each sequence, the minimum free energy in kcal mol−1 was predicted [44 (link)].
RNA sequences were analysed using the RNAfold web server of the Vienna RNA package (
7
DNase I Hypersensitivity Mapping in TGFβ-Treated Cells
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DNase I hypersensitivity mapping was performed according to ref. 98 (link) with brief modifications. NMuMG cells were either vehicle or TGFβ-treated for 2 h. The cells were trypsinized and pelleted before washing and resuspension in buffer A (15 mM Tris-Cl (pH 8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 0.5 mM spermidine, 0.15 mM spermine). Nuclei were extracted by adding buffer A containing NP-40. The nuclei were washed with buffer A and resuspended in pre-warmed lysis buffer at a concentration of 5 × 106/ml and then digested with 75 units of DNase I (Roche) for 5 min at 37 °C. The reactions were terminated by the addition of an equal volume of stop buffer and incubated at 55 °C. After 15 min, proteinase K (final concentration of 20 μg/ml) was added to each digestion reaction and incubated for 16 h at 55 °C. DNA was extracted by careful phenol-chloroform purification using phase-lock gel. DNA fragments of 50–300 bp were selected using low-melting agarose gel and was purified using GenElute Gel Extraction Kit (Sigma-Aldrich). DNA was again purified using MinElute PCR Purification kit and samples were sequenced using Illumina NextSeq 500 system (Illumina) with 75 bp paired-end reads at the Genomic Unit of CABIMER (Sevilla, Spain).
8
Identification of Circular RNA Transcripts
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One microgram of total RNA was used for cDNA synthesis using the iScript Select cDNA Synthesis Kit (Bio-Rad). Outward-facing primers with respect to genomic sequence were designed to specifically amplify the backsplice junctions of representative circRNAs in a PCR assay. PCR reactions were performed for cDNA samples and genomic DNA using 28 cycles. PCR products were analyzed by 2.2% agarose gel electrophoresis and then purified using GenElute Gel Extraction Kit (Sigma-Aldrich). Gel purified PCR products were directly sequenced to identify the gel product.
For real-time PCR, Fast Start Universal SYBR Green Master Mix (Roche) was used to amplify the backsplice junctions using divergent primers; primers for mRNA were obtained from Primer3 software. Each real-time assay was done in triplicate using the Step-One-Plus Real-time PCR system (Life Technologies).
For real-time PCR, Fast Start Universal SYBR Green Master Mix (Roche) was used to amplify the backsplice junctions using divergent primers; primers for mRNA were obtained from Primer3 software. Each real-time assay was done in triplicate using the Step-One-Plus Real-time PCR system (Life Technologies).
9
Cellular Signaling Pathways Modulation
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RPMI 1640 medium (61870-036) and fetal bovine serum [FBS] (10099141) were purchased from Gibco (USA, NY). CORT and neferine (HY-N0441) were purchased from MedChem Express (NJ, USA). The GenElute Gel Extraction Kit (NA1111) was purchased from Sigma-Aldrich (Darmstadt, Germany). BamHI (R0136S) and XhoI (R0146S) were purchased from NEB (NY, USA). Lipofectamine 3000 Transfection Reagent (L3000015) was purchased from Invitrogen (CA, USA). G418 (G8161) was purchased from Solarbio (Beijing, China). RNApure Tissue and Cell Kit (CW0584), HiFiScript cDNA Synthesis Kit (CW2569), and Super TaqMan Mixture (CW2698) were obtained from Cwbiotech (Beijing, China). Antibodies against Bcl-2 (ab32124), Bax (ab32503), Bad (ab32445), p53 (ab26), Bak (ab32371), succinate-CoA ligase GDP-forming beta subunit [SUCLG2] (ab96172), aconitase 2 [ACO2] (ab110321), malate dehydrogenase 1 [MDH1] (ab180152), citrate synthase [CS] (ab96600), isocitrate dehydrogenase [IDH] (ab172964), NF-κB p65 (ab16502), and glyceraldehyde 3-phosphate dehydrogenase [GAPDH] (ab8245) were purchased from Abcam (Massachusetts, US). ELISA kits for IL-1β (ab100562), IL-2 (ab174444), IL-6 (ab46027), TNF-α (ab181421), interferon-γ [IFN-γ] (ab46025), and granulocyte colony-stimulating factor [G-CSF] (ab100524) were purchased from Abcam. Rabbit and mouse secondary antibodies were purchased from ECL.
10
Cloning and Transformation Techniques
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Standard cloning techniques were used to construct plasmids. Escherichia coli DH5α served as the cloning strain, cultured in LB Broth Miller Difco (BD) supplemented with appropriate antibiotics (Ampicillin, 100 μg ml−1, Chloramphenicol, 25 μg ml−1 and Kanamycin, 50 μg ml−1). Enzymes were purchased from New England Biolabs (NEB). Phusion High-Fidelity DNA Polymerase (NEB) was used for PCR amplification. Oligonucleotides used as primers or for annealing were purchased from Microsynth, IDT or Sigma-Aldrich. Digestion products or PCR fragments were purified using GenElute Gel Extraction Kit or Gen Elute PCR Clean Up Kit (both Sigma-Aldrich). Ligations were performed using T4 DNA Ligase (NEB) at 16 °C for 1 h for sticky end overhangs or at 4 °C overnight for blunt-end ligation, followed by transformation into chemically competent cells and plating on LB Agar plates with appropriate antibiotics. Clones were screened by colony-PCR using Quick-Load Taq 2X Master Mix (NEB) or by test restriction. Plasmids were sequenced by Microsynth. Detailed cloning procedure for each plasmid can be found in Supplementary Table 2 , with primers listed in Supplementary Table 3 and gBlocks in Supplementary Table 4 .
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