ddRAD-Seq library was constructed as described by Shirasawa et al. [24 (link)]. A total of 200 ng of genomic DNA for each individual was double-digested with PstI and MspI (FastDigest restriction enzymes; Thermo Fisher Scientific, Waltham, MA, USA), ligated to adapters using the LigaFast Rapid DNA Ligation System (Promega, Madison, WI, USA), and purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to eliminate short (<300 bp) DNA fragments. Purified DNA was diluted with H2O and amplified by 20 cycles of PCR with indexed primers. Amplicons were pooled and separated on a BluePippin 1.5% agarose cassette (Sage Science, Beverly, MA, USA), and fragments of 300–900 bp were purified using the QIAGEN Mini Elute Kit (Qiagen, Hilden, Germany). Then, the library was sequenced using a HiSeq4000 (Illumina, Inc., San Diego, CA, USA).
Ligafast rapid dna ligation system
Manufactured by Promega
Sourced in United States
The LigaFast Rapid DNA Ligation System is a lab equipment product that enables the rapid and efficient ligation of DNA fragments. It facilitates the joining of DNA molecules, a fundamental process in molecular biology and genetic engineering.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq green master mix, by Promega (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Exprep plasmid sv mini, by GeneAll (2 mentions)
Expin gel sv, by GeneAll (2 mentions)
Hispeed plasmid midi kit, by Qiagen (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Minielute kit, by Qiagen (1 mentions)
Agencourt ampure xp, by Beckman Coulter (1 mentions)
Fastdigest restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Zyppy plasmid miniprep kit, by Zymo Research (1 mentions)
Quick dna miniprep kit, by Zymo Research (1 mentions)
Pgl2 basic vector, by Promega (1 mentions)
Pnf kb luc, by Takara Bio (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
High pure plasmid isolation kit, by Roche (1 mentions)
Minelute kit, by Qiagen (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
14 protocols using ligafast rapid dna ligation system
1
ddRAD-Seq Library Construction Protocol
2
Bisulfite Sequencing of iPSC and PHDF Lines
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DNA from 8 iPSC clones and 4 PHDF lines was isolated and bisulfite-treated as described [8 (link)]. DNA from 8 iPSC clones and 4 PHDF cell lines was isolated with a Quick-DNA Miniprep Kit (#D3025, Zymo Research, Irvine, CA, USA). Then, 1.5 µg DNA was bisulfite converted with EZ DNA Methylation Kit (#D5001, Zymo Research, Irvine, CA, USA), according to the manufacturer’s protocol. OCT3/4 and NANOG promoter was amplified with 500 nM specific primers (listed in Table S2 ) and the GoTaq Green Master Mix (#M7122, Promega, Madison, WI, USA). PCR was performed with the following thermal profile: 95 °C/2 min, 42 cycles of 95 °C/30 s + 61 °C/30 s + 72 °C/30 s, and a final extension at 72 °C for 7 min. Amplified fragments of OCT3/4 and NANOG promoters were ligated to pGEM-T Easy vector (#A1360, Promega, Madison, WI, USA) with a LigaFast Rapid DNA Ligation System (#M8221, Promega, Madison, WI, USA) and cloned into E. coli DH5a (#T3007, Zymo Research, Irvine, CA, USA). The plasmids from individual clones were purified with a Zyppy™ Plasmid Miniprep Kit (#D4036, Zymo Research, Irvine, CA, USA), sequenced (Genomed, Warsaw, Poland), and analyzed with the BISMA application (RRID:SCR_000688, Jacobs University Bremen; Germany) [33 (link)]. The percentage of methylation (MtI) was calculated according to the formula: MtI% = Cm/(Cm + Cnm).
3
Construction of Luciferase Plasmids with NF-kappaB Sites
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For the construction of the luciferase plasmids, four copies of each kappaB site or mutant site (see below) were ligated (LigaFast rapid DNA ligation System, Promega, WI, USA) into a BglII and MluI-digested pGL2-Basic vector (Promega). The insert was generated by annealing a synthetic 5' phosphorylated oligonucleotides (Syntezza, Jerusalem, Israel). A control plasmid containing the consensus NF-kappaB sequence linked to the luciferase sequence was obtained from Clontech (pNF-kB-Luc, CA, USA). The CMVp65 and CMVdeltaNIkappaB plasmids have been previously described [35] .
The sequences of the kappaB sites inserted into the luciferase vector are as follows:
MGMT-kB1-Luc: 4XGTAAAGTCCCC
MGMT-Mut-kB1-Luc: 4XGTAAAGTCGGC
MGMT-kB2-Luc: 4XGGAACACCCC
MGMT-Mut-kB2-Luc: 4XGTAAAGTCGGC
The sequences of the kappaB sites inserted into the luciferase vector are as follows:
MGMT-kB1-Luc: 4XGTAAAGTCCCC
MGMT-Mut-kB1-Luc: 4XGTAAAGTCGGC
MGMT-kB2-Luc: 4XGGAACACCCC
MGMT-Mut-kB2-Luc: 4XGTAAAGTCGGC
4
Generating V2 Deletion Plasmids
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To create the V2 deletion plasmids, the pGL4.11 vector cloned with the V2 original construct was amplified by PCR using 5′-phosphorylated primers; S, 5′-GGC ACA CAG AGA TAC GCG CA-3′, and AS, 5′-TGT GTG CGT GTA GGG GGT TA-3′ (Supplementary Fig. 4 ). PCR was performed with 12.5 µl of SmartGene 2 × pfu Mixed Taq Advanced (SJ Bioscience, Republic of Korea), 9.5 µl of nuclease-free water, 1 µl of primers (10 pmol/µl), and 1 µl of pGL4.11 vector cloned with V2 original construct as template DNA. The PCR conditions were as follows: initialization at 94 °C for 3 min, 22 thermal cycles of 94 °C for 40 s, primer-specific annealing at 61 °C for 30 s, 72 °C for 6 min, and a final elongation step at 72 °C for 5 min.
All PCR products were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a pGL4.11-T vector (Promega, USA) and ligated by LigaFast Rapid DNA Ligation System (Promega, USA). Plasmid isolation was performed with the Exprep Plasmid SV mini (GeneAll, Republic of Korea). The vector into which the PCR products were inserted was verified by colony PCR.
All PCR products were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a pGL4.11-T vector (Promega, USA) and ligated by LigaFast Rapid DNA Ligation System (Promega, USA). Plasmid isolation was performed with the Exprep Plasmid SV mini (GeneAll, Republic of Korea). The vector into which the PCR products were inserted was verified by colony PCR.
5
Pax3 Splice Variant Misexpression
6
Phylogenetic Analysis of Viral RdRp Gene
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The RdRp PCR products were gel purified using the NucleoSpin® Gel and PCR Clean-up kit (MACHEREY-NAGEL GmbH & Co. KG), and sequenced directly using an automated ABI PRISM 377 DNA sequencer. When multi peaks were shown in chromatogram at same position from direct sequencing, PCR products were cloned using the pGEM®-T Easy Vector System and the LigaFast™ Rapid DNA Ligation System (Promega) before sequencing. Five colonies were picked up for sequencing. Sequences were cleaned using Bio-edit program and aligned with reference sequences collected from GenBank. Alignments were performed using Multiple Alignment using Fast Fourier Transform (MAFFT) [26 (link)]. Phylogenetic trees were created based on 357 and 299 bp RdRp gene sequence using the maximum likelihood method. Bootstrap values were determined using 1000 replicates via RaxmlGUI 1.3 with outgroup (Bulbul CoV/HKU11–934/Pycnonotus jocosus/CHN/2007/FJ376619) using the GTRI substitution model [27 (link)]. The phylogenetic tree was visualised using the FigTree program, version 1.4.2 [28 ].
7
Standard DNA Cloning and Purification
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Standard protocols were used for cloning and transformations. All restriction endonucleases and DNA modification enzymes were purchased from New Englands Biolabs. Polymerase chain reactions (PCRs) were performed with PrimeSTAR™HS DNA Polymerase from Takara. DNA ligations were performed with LigaFast™ Rapid DNA Ligation System from Promega. The High Pure Plasmid Isolation kit from Roche was used to purify plasmidic DNA. Chromosomal DNA was purified using the Wizard Genomic DNA purification kit from Promega. DNA fragments and plasmids were excised or purified using the MinElute kits from Qiagen.
8
Genetic manipulation of P. thermoglucosidasius
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Plasmids and primers used in this study are listed in Supplementary Tables S3,S4 . All primers were designed using ApE (University of Utah) and analysed with OligoAnalyzer Tool (IdtDNA). For the amplification of the desired DNA products, Phusion High-fidelity DNA polymerase was used for cloning purpose and dream Tag Green PCR (Thermo Fisher Scientific, United Kingdom) master mix was used for strain verification. DNA fragments that had undergone restriction digestions were purified using Zymoclean™ Gel DNA Recovery Kit (ZymoResearch, United Kingdom) before ligation using LigaFast™ Rapid DNA Ligation System (Promega). Chemically competent E. coli Top10 cells produced in-house were used to propagate the constructed plasmids and plasmids were harvested using Monarch® Plasmid Miniprep Kit (New England BioLabs). All cloning procedures were conducted following the manufacturer’s instructions. Sanger sequencing (Source Biosciences; EurofinsDNA) were routinely performed to confirm the authenticity of plasmid constructs. Preparation of P. thermoglucosidasius electro-competent cells and electroporation procedure using Genepulser electroporator (BioRad, United Kingdom) was undertaken in accordance with the method previously described (Sheng et al., 2017 (link)).
9
Genomic DNA Extraction and PCR Amplification
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The DNeasy Blood & Tissue Kit (Qiagen, Germany) was used to extract genomic DNA (gDNA) from human cell line HEK293A. gDNA was used for PCR amplification with 2 × TOP simple DyeMix (aliquot)-HOT premix (Enzynomics, Republic of Korea). The PCR mixture contained with 10 µL of contained with 2 × TOPsimple DyeMix, 7 µL of distilled water, 1 µL of gDNA template, and 1 µL of each primers (10 pmol/µL). The condition of PCR were as follow: an initialization at 95 °C for 5 min, 35 thermal cycles of 94 °C for 40 s, primer-specific annealing temperatures for 40 s, 72 °C for 40 s, and a final elongation step at 72 °C for 5 min. The products of PCR were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a psi-CHECK2 vector (Promega, USA) by LigaFast Rapid DNA Ligation System (Promega, USA). The Exprep Plasmid SV, mini (GeneAll, Republic of Korea) was used for plasmid isolation.
10
Generating Transgenic Arabidopsis Thaliana
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Genomic DNA was extracted from the leaves of A. thaliana transformants #9, 15, 36, 69, 77, 81, 106, 186, 189, 224, 227, and 253. Fragments of the mutated AlSRKb genes from the transformants were amplified by PCR using the primer pair AtS1(−28)/AlSRKb(1260)R (Supplemental Data Set 13 ). The amplified fragments were introduced into the KpnI/SacI sites of AtS1pro:AlSRKb FLAG+AlSCRb using the LigaFast Rapid DNA Ligation System (Promega). The mutations N89D, M170T, and G173D were introduced into the AlSRKb sequence by recombinant PCR-mediated site-directed mutagenesis using AtS1pro:AlSRKb FLAG+AlSCRb as a template and the appropriate sequence-specific primer pair: (AlSRKb(N89D)F/AlSRKb(N89D)R for AlSRKb(N89D); (AlSRKb(M170T)F/AlSRKb(M170T)R for AlSRKb(M170T); and AlSRKb(G173D)F/AlSRKb(G173D)R for AlSRKb(G173D) (Supplemental Data Set 13 ).
The resulting plasmids were sequenced at Eurofins Genomics KK (Tokyo, Japan) to confirm the absence of PCR-generated polymorphisms. The sequence-verified plasmids were used to transform Agrobacterium tumefaciens strain GV3101 (Koncz & Schell, 1986 (link)) and introduced into wild-type C24 plants by the floral dip method (Clough and Bent, 1998 (link)). Transformants were selected on Murashige and Skoog medium containing 50 mg/mL hygromycin.
The resulting plasmids were sequenced at Eurofins Genomics KK (Tokyo, Japan) to confirm the absence of PCR-generated polymorphisms. The sequence-verified plasmids were used to transform Agrobacterium tumefaciens strain GV3101 (Koncz & Schell, 1986 (link)) and introduced into wild-type C24 plants by the floral dip method (Clough and Bent, 1998 (link)). Transformants were selected on Murashige and Skoog medium containing 50 mg/mL hygromycin.
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