Total complete RNA was isolated from in vitro-grown Xushu55-2 plants and then reverse transcribed to generate cDNA (Zhou et al., 2019 (link)). Based on the expressed sequence tag (EST) selected from the transcriptome sequencing data of Xushu55-2 (Zhu et al., 2018 (link)), the 5′-untranslated region (UTR) and 3′-UTR of IbBT4 were amplified using rapid amplification of cDNA ends (RACE) procedure using 5′- Full RACE Kit and 3′-Full RACE Core Set Ver.2.0 Kit (TaKaRa, Beijing, China). The cDNA sequence was analyzed by NCBI1 . The coding sequence (CDS) was cloned by PCR with specific primers. Its genomic sequence was cloned from Xushu55-2 genomic DNA via PCR in conjunction with specific primers. The promoter region was cloned with Universal Genome Walker 2.0 Kit (TaKaRa, Dalian, China). All of the specific primers are listed in Supplementary Table S1 . IbBT4 was annotated in the NCBI databases2 . Amino acid sequence alignments, phylogenetic relationships and exon-intron structure were analyzed with DNAMAN software, MEGA 7.0 software and the Splign tool, respectively, and the molecular weight and theoretical isoelectric point (pI) were calculated via online software (Kang et al., 2019 (link)). The cis-acting regulatory elements in the promoter region were analyzed via the PlantCARE database.
3 full race core set ver 2.0 kit
Manufactured by Takara Bio
Sourced in China, Japan
The 3'-Full RACE Core Set Ver.2.0 kit is a laboratory equipment product designed for 3' Rapid Amplification of cDNA Ends (3' RACE) experiments. It contains the essential components required to perform this technique, which is used to determine the 3' end of a transcript.
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Lab products found in correlation
5 full race kit, by Takara Bio (9 mentions)
Rnaiso plus reagent, by Takara Bio (6 mentions)
Pmd18 t vector, by Takara Bio (5 mentions)
M mlv reverse transcriptase kit, by Takara Bio (2 mentions)
Gel extraction kit, by Sangon (2 mentions)
Universal genome walker 2.0 kit, by Takara Bio (1 mentions)
Gel extraction kit, by CWBIO (1 mentions)
Genome walking kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Bcabest rna pcr kit, by Takara Bio (1 mentions)
Peasy t1 vector, by Transgene (1 mentions)
Taq pcr mastermix, by Tiangen Biotech (1 mentions)
Dna marker, by Tiangen Biotech (1 mentions)
N n dicyclohexylcarbodiimide dcc, by J&K Scientific (1 mentions)
N fmoc amino acids, by GL Biochem (1 mentions)
Methanol, by J&K Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Easyspin plant rna extraction kit, by Aidlab (1 mentions)
M mlv kit, by Takara Bio (1 mentions)
13 protocols using 3 full race core set ver 2.0 kit
1
Cloning and Characterization of IbBT4 from Sweet Potato
2
Cloning and Characterization of Sugarcane R2R3-MYB Transcription Factor
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A sugarcane R2R3-MYB EST was obtained based on the bioinformatics analysis using the data from the previous RNA-Seq experiments (not yet published). Two nested gene-specific 3′ RACE primers was designed according to the EST sequence information, and one pair of gene-specific primer was designed to amplify the given target regions using genomic DNA as a template. Primer sequences are as follows:
3′ RACE GSP1: 5′-ACATAGTGGCTTCTTCTCCC-3′;
3′ RACE GSP2: 5′-TATCCAAGGTAGAAGCGAGCAA-3′;
ScMyb2 F: 5′-TATCCAAGGTAGAAGCGAGCAA-3′ (same to 3′ RACE GSP2);
ScMyb2 R: 5′-CCATAAGCATACCTCCAGTGTT-3′.
The method used in 3′ RACE was followed to the specifications of the 3′-Full RACE Core Set Ver.2.0 Kit (Takara). A full-length R2R3-MYB homolog gene of sugarcane (named ScMYB2) was identified by Blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) with two Myb-like DNA-binding domains (pfam00249). The open reading frame (ORF) of the full-length cDNA sequence of ScMYB2 was predicted using the ORF Finder online tool from NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html ). Sequence alignment was performed using DNAMAN 5.2.2 software.
3′ RACE GSP1: 5′-ACATAGTGGCTTCTTCTCCC-3′;
3′ RACE GSP2: 5′-TATCCAAGGTAGAAGCGAGCAA-3′;
ScMyb2 F: 5′-TATCCAAGGTAGAAGCGAGCAA-3′ (same to 3′ RACE GSP2);
ScMyb2 R: 5′-CCATAAGCATACCTCCAGTGTT-3′.
The method used in 3′ RACE was followed to the specifications of the 3′-Full RACE Core Set Ver.2.0 Kit (Takara). A full-length R2R3-MYB homolog gene of sugarcane (named ScMYB2) was identified by Blastx (
3
Cloning of Hepatopancreas GST Genes
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RNAiso Plus Reagent (TaKaRa, Kusatsu, Japan) was used to isolate total RNA from hepatopancreas tissue according to the manufacturer’s protocol. The reverse transcriptase M-MLV Kit (TaKaRa, Kusatsu, Japan) was used to synthesize first-strand cDNA, and 3′-rapid amplification of cDNA ends (RACE) was performed using a 3′-full RACE Core Set Ver. 2.0 Kit (TaKaRa, Kusatsu, Japan) to determine the 3′ ends of MnGST-1 and MnGST-2. All primers used for cloning are listed in Table 1 . Polymerase chain reaction (PCR) products were purified using a gel extraction kit (CWBIO, Beijing, China) and sequenced using an ABI3730 DNA analyzer (ABI, Tampa, FL, USA) after insertion into the PMD-18T vector (TaKaRa, Kusatsu, Japan).
4
Cloning and Sequencing of MnLDH Gene
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A 3′-full RACE Core Set Ver. 2.0 kit and a 5′-full RACE kit (TaKaRa, Dalian, China) were used to determine the cDNA 3′- and 5′-ends of the MnLDH gene. The primers used in this cloning are listed in Table 1 . PCR products were purified using a gel extraction kit (Sangon, Shanghai, China), followed by sequencing using an ABI3730 DNA analyzer. The 5ʹ- promoter region of MnLDH was cloned using a genome walking kit (TaKaRa, Dalian, China) with primers GSPR1, GSPR2, GSPR3 by previous methods [43 (link)]. A 2017-bp DNA fragment was obtained and cloned into vector pMD18-T (Takara, Dalian, China) for sequencing. Nucleotide sequence and bioinformatic analyses were performed as described previously [44 (link)].
5
Isolation and Characterization of TsGPX Genes
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We searched the T. salsuginea ESTs in the GenBank using the CDs of GPX1-8 from A. thaliana. The ESTs representing TsGPX genes were assembled using software DNASTAR (DNASTAR Inc., Madison, WI, USA). The primers used for isolating full length CDs of TsGPX gene were designed using Primer3 (http://primer3.ut.ee/ ). Total RNA from T. salsuginea seedlings was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The potential contaminating genomic DNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA). The cDNA was synthesized using BcaBEST RNA PCR Kit (Takara, Shiga, Japan) according to the manufacturer’s protocol. 3′ RACE were performed to isolate full length CDs of some members of TsGPX gene family using 3′ Full RACE Core Set Ver.2.0 kit (TaKaRa, Shiga, Japan). All primers are listed in Tables S2 and S3 .
6
Cloning and Validation of Mn-Plk1 Gene
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Table 1 http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) based on the open reading frame (ORF) of Mn-Plk1. The full-length cDNA of Mn-Plk1 was cloned using the RACE technique following the methods described in previous studies (39 (link), 40 (link)). Briefly, the total RNA from the testis was extracted using RNAiso Plus Reagent (Takara, Shiga, Japan), and the extracted total RNA was then used to synthesize the templates for 3′cDNA and 5′cDNA cloning using the 3′-Full RACE Core Set Ver.2.0 kit and the 5′-Full RACE kit (Takara), respectively. After the assembling, two pairs of primer were designed based on the full-length cDNA sequence of Mn-Plk1, in order to verify the accuracy of Mn-Plk1 cDNA sequence (Table 1
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7
Cloning and Sequencing of Crustacean EcR and RXR
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Two partial cDNA sequences highly similar to published EcRs and RXRs (cDNA in Genbank) from our deep sequencing data were identified, respectively. Based on these two partial cDNA sequences, gene-specific 3′ and 5′ primers were designed for RACE PCR (rapid amplification of cDNA ends) (Table 1 ). 3′ and 5′ RACE cDNA were prepared from total RNA of P. clarkii (hepatopancreas), using a 3′-Full RACE Core Set Ver.2.0 Kit and 5′-Full RACE kit (Takara, Dalian, China) according to the manufacturer’s instructions, respectively. After performing two rounds of PCR to obtain 3′ and 5′ end fragments of PcEcR and PcRXR, the final PCR products were cloned into the pEASY-T1 vector (Transgen, Beijing, China). The recombinant plasmids were used to transform E. coli (Escherichia coli) TOP 10 competent cells, isolated, and sequenced.
8
Androgenic Gland RNA Extraction and Cloning
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As described in detail previously [12 (link),13 (link)], total androgenic gland RNA was extracted using RNAiso Plus Reagent (Takara Bio Inc., Dalian, China). The templates for 3′cDNA and 5′cDNA cloning were synthesized by using the 3′-Full RACE Core Set Ver.2.0 kit (Takara Bio Inc., Dalian, China) and the 5′-Full RACE kit (Takara Bio Inc., Dalian, China). The specific primers used for genes cloning were listed in Table 2 . The BLASTX and BLASTN search program (http://www.ncbi. nlm.nih.gov/BLAST/ ) and the ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html ) were employed to analyze the structural characteristics. MEGA X (https://www.megasoftware.net ) was used to construct the phylogenetic trees, followed by the maximum-likelihood method with Bootstrap method of 1000 replications.
9
Peptide Synthesis and Animal Experiments
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The pGEM-T Easy Vector System, E. coli. Dh5α, 2×Taq PCR Master Mix, and DNA Marker were obtained from TIANGEN Biotech Co., Ltd. (Beijing, China). The 3′-Full RACE Core Set Ver.2.0 kit was obtained from TaKaRa (Dalian, China). The Nucleic Acid purification kit was purchased from Dongsheng Biotech (Guangzhou, China). N-Fmoc-amino acids and HOBt were sourced from GL Biochem Ltd. (Shanghai, China). Rink resin was purchased from Tianjin Nankai Hecheng S&T Co. (Tianjin, China). N,N′-Dicyclohexylcarbodiimide (DCC) and Methanol were purchased from J&K Chemical Ltd. (Shanghai, China) and Honeywell Burdick & Jackson (Muskegon, MI United States), respectively. The other reagents were of analytical grade.
Adult male Sprague-Dawley rats (SD, 250 g, Beijing Animal Center, China) were housed in groups of eight and maintained on a 12 h light-dark cycle (light cycle from 8 am to 8 pm) at a temperature of 23 ± 2°C and relative humidity of 50%. Food pellets and water were available ad libitum. All experiments were conducted in accordance with the guidelines of the Beijing Institutes for Biological Sciences Animal Research Advisory Committee and conformed to the European Community directives for the care and use of laboratory animals.
Adult male Sprague-Dawley rats (SD, 250 g, Beijing Animal Center, China) were housed in groups of eight and maintained on a 12 h light-dark cycle (light cycle from 8 am to 8 pm) at a temperature of 23 ± 2°C and relative humidity of 50%. Food pellets and water were available ad libitum. All experiments were conducted in accordance with the guidelines of the Beijing Institutes for Biological Sciences Animal Research Advisory Committee and conformed to the European Community directives for the care and use of laboratory animals.
10
Isolation and Characterization of PI Homolog in M. wufengensis
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Total RNA of M. wufengensis were extracted using EASYspin plant RNA Extraction Kit (Aidlab, Beijing) according to the manufacturer’s instructions. And for each sample, 1 μg RNA was used for cDNA synthesis with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, United Ststes) as previously described (Jing et al., 2014 (link), 2015 (link)). A 659 bp 3′ end of PI homolog from M. wufengensis was amplified from the cDNA through 3′ RACE-specific primer GSPPI and 3′ RACE Outer Primer by the 3′-full RACE Core Set Ver.2.0 kit (TaKaRa, Japan), and a 910 bp 5′ end was amplified through 5′ RACE-specific primer PIGSP1 and PIGSP2 by 5′-full RACE Kit (TaKaRa, Japan). After assembly, the integrity of the cDNA sequences of the PI homolog from M. wufengensis were then verified using the forward primer MawuPIF and the reverse primer MawuPIR. PCR was performed with a 5 min at 94°C denaturation step, followed by 30 cycles of 45 s at 94°C, 45 s annealing at 56°C, 45 s extending at 72°C, with a final extension period of 10 min at 72°C. These PCR products were subcloned into pMD18-T Vector (TaKaRa, Japan) and sequenced. The primers used for isolation of PI homolog in M. wufengensis were listed in Supplementary Table S1 .
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