The resulting embryogenic calluses of G. raimondii and G. sturtianum were treated with 200 mM NaCl after they were grown in culture for 21 days. Three biological replicates were harvested for each sample at various timepoints (0, 6, 12, 24, and 48 h) after exposure to salt stress. Total RNA of each sample was isolated using a modified CTAB method according to the instruction manual (Zhao et al., 2012 (link)), and 1 μg RNA per sample was used as input material for cDNA synthesis. The first-strand cDNA synthesis was accomplished using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H−); then the second-strand synthesis was accomplished using DNA polymerase I and RNase H to generate the final cDNAs. These cDNAs were subjected to end-repair/dA-tail (DNA A-Tailing Kit, TaKaRa) and adaptor ligation, and the resulting libraries were sequenced on the Illumina Novaseq platform as 150-bp paired-end reads with 6 × depth.
Dna a tailing kit
Manufactured by Takara Bio
Sourced in China
The DNA A-Tailing Kit is a laboratory product designed to add a single adenine (A) nucleotide to the 3' end of DNA fragments. This process, known as 'A-tailing', is a common technique used in molecular biology workflows to facilitate the subsequent ligation of DNA fragments into plasmid vectors.
Automatically generated - may contain errors
Lab products found in correlation
Pmd19 t vector, by Takara Bio (5 mentions)
Pmd18 t vector, by Takara Bio (3 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Primerstar max dna polymerase, by Takara Bio (2 mentions)
M mlv reverse transcriptase, by Takara Bio (2 mentions)
Novaseq platform, by Illumina (1 mentions)
Pmd20 t, by Takara Bio (1 mentions)
Abi prism 377xl dna sequencer, by Thermo Fisher Scientific (1 mentions)
Kod onetm pcr master mix, by Toyobo (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Pmd18 t, by Takara Bio (1 mentions)
Dnase 1, by Promega (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Random primer, by Takara Bio (1 mentions)
Platinum superfi 2 high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
E z n a viral rna kit, by Omega Bio-Tek (1 mentions)
Gel doc ez imaging system, by Bio-Rad (1 mentions)
Golden view, by Takara Bio (1 mentions)
13 protocols using dna a tailing kit
1
Transcriptome Analysis of Salt-Stressed Cotton
2
Cloning and Sequencing of Tyrosine Phosphatase
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The PCR-amplified DNA was recovered from the gel using the Agarose Gel DNA Purification kit v2.0 (cat. no. DV805; Takara Biotechnology Co., Ltd.) and a poly-A tail was added using the DNA A-Tailing Kit (cat. no. 6109; Takara Biotechnology Co., Ltd.). The poly-A tailed product was cloned into the simple vector pMD20-T (cat. no. D107A; Takara Biotechnology Co., Ltd. according to the manufacturer's instructions. Competent Escherichia coli (E. coli) JM109 cells (cat. no. 9052; Takara Biotechnology Co., Ltd.) were transformed with the recombinant plasmid pMD20T-Tyr p 13 using the heat-shock method. Positive clones were selected by blue/white screening on Luria-Bertani (LB) plates containing 100 µg/ml ampicillin and samples were sequenced using Bca BEST sequencing primer RV-M/M13-47 (Takara Biotechnology Co., Ltd.) on an ABI PRISM 377XL DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.).
3
CYP2B6 Gene Sequencing and Haplotype Analysis
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For nonsynonymous variants, the entire CYP2B6 (30,957 bp) gene was first amplified with KOD OneTM PCR Master Mix (TOYOBO, Japan) and then purified with 0.6 × AMPure XP beads (Beckman Coulter, High Wycombe, UK). A DNA A-tailing Kit (Takara, Japan) was used for dA-tailing for 20 min at 72 °C. The A-tailed DNA amplicons were purified by 0.6 × AMPure XP beads and then ligated to barcodes (EXP-NBD104, ONT, UK). After purification, adapters were ligated to the DNA ends with the SQK-LSK110 ligation kit (ONT, Oxford, UK) and the samples were purified again with 0.6 × volume of AMPure XP beads. The adapted library was quantified with a Qubit fluorometer and 50–100 fmol DNA library was loaded onto a GridION flow-cell (R9.4.1) according to manufacturer instructions and run for 17.5 h. The raw sequencing data was base called using the MinKNOW software in default mode. The reads, in fastq format, were mapped to the human reference genome GRCh38 using Minimap2. The alignment data were transferred to bam format and sorted using samtools. The sorted and indexed bam data were viewed with IGV_2.14.0 and the haplotype of each SNP was determined manually (Supplementary Fig. 1 ). The novel haplotypes were submitted to DNA Data Bank of Japan and PharmVar for nomenclature.
4
Cloning and Promoter Analysis of Key Tomato Genes
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Total RNA was isolated from each sample using the RNAiso Plus (Takara, Dalian, China) following the manufacturer’s protocol. In the reverse transcription, 1 μg total RNA samples were used to synthesize the first-strand cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA) with oligo(dT)20 primer.
To clone the full length of SlMBP21 and SlMADS1, 1–2 μL cDNA was used to employ the PCR reactions with primers SlMBP21-Full-F/-R and SlMADS1-Full-F/-R, respectively (Table S1 ). After being tailed with a DNA A-Tailing kit (Takara, Dalian, China), the amplified products of SlMBP21 and SlMADS1 were linked into the vector pMD18-T (Takara, Dalian, China) to obtain SlMBP21-pMD18-T and SlMADS1-pMD18-T, respectively. Positive clones of each vector were picked out through Escherichia coli DH5α transformation and were conformed by sequencing.
To analyze putative cis-elements in the SlMBP21 and SlMADS1 promoter region, promoter analysis was carried out using the promoter sequences (2800 bp regions upstream initiation codon ATG of gene-predicted ORF) of each gene on the Plant CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ , accessed on 3 July 2023).
To clone the full length of SlMBP21 and SlMADS1, 1–2 μL cDNA was used to employ the PCR reactions with primers SlMBP21-Full-F/-R and SlMADS1-Full-F/-R, respectively (
To analyze putative cis-elements in the SlMBP21 and SlMADS1 promoter region, promoter analysis was carried out using the promoter sequences (2800 bp regions upstream initiation codon ATG of gene-predicted ORF) of each gene on the Plant CARE website (
5
Cloning and Characterization of SlCMB1 Gene
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The total RNA from all WT and transgenic tomato plants tissues was extracted using the RNAiso Plus reagent (Takara, China) following the instructions of manufacturer. In order to synthesize the first strand cDNA, 1 μg total RNA samples which were digested with the Dnase I (Promega, USA) was used to perform the reverse transcription using the M-MLV reverse transcriptase (Promega) with the oligo(dT)20 primer according to the manufacturer’s protocol.
The full length of SlCMB1 gene was cloned using 1–2 μL cDNA with primers SlCMB1-Full-F and SlCMB1-Full-R (Supplementary TableS1 ). The DNA A-Tailing kit (Takara) was used to tail the amplified products. After that, the tailed products were cloned into the pMD18-T vector (Takara). The Escherichia coli JM109 transformation was performed to pick out the positive clones and confirmed by sequencing. Multiple sequence alignment was performed for comparison with other MADS-box proteins by DNAMAN (Version 6.0). The phylogenetic tree was constructed by MEGA(Version 5.2) according to the neighbor-joining bootstrap method as follows: bootstrap analysis of 1,000 replicates, pairwise deletion and poisson model. Moreover, 3500 bp nucleotide sequence upstream of the initiation codon ATG of predicted ORF of SlCMB1 gene was used to perform the promoter analysis on the Plant CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ).
The full length of SlCMB1 gene was cloned using 1–2 μL cDNA with primers SlCMB1-Full-F and SlCMB1-Full-R (Supplementary Table
6
Cloning and Sequencing of Resistance Genes
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The primers used to clone candidate genes with potential upstream promoter regions are shown in Table 2 . PrimeSTAR HS DNA Polymerase (TaKaRa, Dalian, China) was used to amplify resistance genes according to the manufacturer's instructions. A poly(A) tail was added to each purified PCR product (prom-ORF) using the DNA A-Tailing Kit (TaKaRa, Dalian, China), and the fragment was then cloned into the pMD™19-T vector (TaKaRa, Dalian, China). The resulting recombinant plasmid (pMD™19-T-prom-ORF) was transformed into E. coli DH5α using the calcium chloride method. Transformants were selected on LB agar plates containing 100 μg/mL ampicillin. The cloned PCR product was further confirmed by Sanger sequencing.
7
Cloning and sequencing of SpHsp27
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According to the transcriptome sequencing of S. prenanti, the specific Hsp27 primers were designed (Table 1 , Hsp27-F and Hsp27-R) and synthetized by Tsingke Biotechnology Co., Ltd. (Xi’an, China). Liver cDNA was used as a template for SpHsp27 amplification, with Primerstar® Max DNA polymerase (TaKaRa, Dalian, China). The PCR program was as follows: 35 cycles at 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 40 s. The PCR products were tailed A with DNA A-Tailing Kit (TaKaRa, Dalian, China). The obtained PCR products were ligated into a pMD19-T vector (TaKaRa, Dalian, China), and transformed into competent Escherichia coli DH5α cells. The positive bacteria clones were sequenced.
8
Full-Length Genomic Amplification of Swine Coronaviruses
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According to the instruction manual, viral RNA was extracted using the E.Z.N.A Viral RNA Kit (OMEGA Bio-Tek). The cDNA was synthesized using Reverse Transcriptase M-MLV (Takara, Dalian, China) and Random Primer (Takara, Dalian, China). We designed 12 and 10 pairs of specific primers for full-length genomic amplification of TGEV and PDCoV positive samples, respectively (Tables S1 and S2 ), by Platinum SuperFi II high-fidelity DNA polymerase (Invitrogen, Waltham, MA, USA). PCR amplification products were ligated using DNA A-Tailing Kit (Takara, Dalian, China) and pMD18-T vectors (Takara, Dalian, China), and finally, all plasmids were sent to Tsingke Biotechnology Co., Ltd. (Beijing, China). for Sanger sequencing.
9
Gel Electrophoresis and DNA Sequencing Protocol
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To separate the indicated DNA fragments, the prepared PCR products were added to a 1% agarose gel with Golden View™ (TaKaRa) and electrophoresed with DL2000 DNA marker (TaKaRa) at 120 V for approximately 25 min, as we reported (20 (link)). PCR band products were visualized using a Gel Doc™ EZ imaging system (Bio-Rad, CA). Positive PCR products were purified (MiniBEST Agarose Gel DNA Extraction Kit, TaKaRa), tailed with “A”-overhang tails (DNA A-Tailing Kit, TaKaRa), and cloned into the pMD19-T vector (TaKaRa). Positive recombinant plasmids were sequenced by Ruibiotech Co., Ltd. (Beijing, China). Nucleotide sequences were blasted against sequences deposited in GenBank using the basic local alignment search tool, nt (BLASTn)1 for similarity analysis.
10
Porcine Serum and Tissue miRNA Analysis
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Total RNA from porcine serum and tissues was obtained using Trizol Reagent or Trizol LS Reagent. Small RNA was reverse-transcribed to cDNA and stem-loop PCR was performed using Bulge-LoopTM miRNA qRT-PCR Starter Kit (RiboBio); the A-tailing was added using DNA A-Tailing Kit (Takara). Next, the products were cloned into the pMD19-T vector (Takara) and around 40 monoclonals were randomly picked for each miRNA. Then, Sanger sequencing with the M13 primer was used to determine the sequences of the PCR products.
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