RNAs from different nodule developmental stages for Iso-Seq were used for PCR validation. cDNAs were synthesized by reverse transcriptions with Reverse Transcriptase M-MLV (RNase H-) (cat. no.639575, TaKaRa, Shiga, Japan, http://www.takarabiomed.com.cn , accessed on 4 June 2022). Two micrograms total of RNA was used for the synthesis of the first-strand cDNA, and then the validation of different isoforms and lncRNAs from Pacbio sequences was carried out. PCR amplification was performed with the designed specific primers by 2 × Taq Plus Master Mix (Dye Plus) (P212-01, Vazyme, Nanjing, China, http://www.vazyme.com , accessed on 4 June 2022). Soybean elongation factor 1-alpha (TEFS1 gene, JD017G0182700) was used as the control. All specific primers used for PCR were listed in Table S2 . For each RT-PCR experiment, three biological replicates were conducted.
Taq plus master mix dye plus
Manufactured by Vazyme
Sourced in China
2 × Taq Plus Master Mix (Dye Plus) is a ready-to-use solution containing Taq DNA polymerase, dNTPs, and necessary reaction buffer components. It includes a tracking dye for direct gel loading.
Automatically generated - may contain errors
Lab products found in correlation
Primestar max dna polymerase, by Takara Bio (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Oligonucleotide primers, by Tsingke (1 mentions)
Kod plus kit, by Toyobo (1 mentions)
Ni2 nta superflow resin, by Qiagen (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Beyotime (1 mentions)
14c thr, by PerkinElmer (1 mentions)
14c ser, by PerkinElmer (1 mentions)
T4 polynucleotide kinase, by Vazyme (1 mentions)
Anti c myc magnetic beads, by MedChemExpress (1 mentions)
Trelief prestained protein ladder, by Tsingke (1 mentions)
Ampicillin sodium, by MedChemExpress (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
Lipo8000 transfection reagent, by Beyotime (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
4 protocols using taq plus master mix dye plus
1
PCR Validation of Soybean Isoforms and lncRNAs
2
Protein Expression and Purification Protocol
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T4 DNA ligase and Lipo8000 transfection reagent were purchased from Beyotime Biotechnology (Shanghai, China). 2 × Taq Plus Master Mix (Dye Plus) and T4 polynucleotide kinase was purchased from Vazyme Biotechnology (Nanjing, China). The KOD-Plus Mutagenesis Kit and KOD-Plus Kit were from TOYOBO (Osaka, Japan). Ni2+-NTA Superflow resin was purchased from Qiagen Inc. (Hilden, Germany). Dynabeads Protein G, 4,6-diamidino-2-phenylindole (DAPI), MitoTracker, Pierce Silver Stain kit, and Alexa Fluor 488-conjugated secondary antibody were obtained from Thermo Scientific (Waltham, MA, USA). [14C]Thr, [14C]Ser and [14C]Tyr were obtained from Perkin Elmer Inc. (Waltham, MA, USA). PrimeScript RT Master Mix and PrimeSTAR Max DNA Polymerase were obtained from TaKaRa (Kyoto, Japan). Trelief Prestained Protein Ladder and oligonucleotide primers were obtained from Tsingke (Shanghai, China). Competent E. coli BL21(DE3) were purchased from Weidi Biotechnology (Shanghai, China). Serum-free Cell Cryopreservation Medium was obtained from Epizyme Biomedical Technology (Shanghai, China). Anti-c-Myc magnetic beads, kanamycin sulfate and ampicillin sodium were purchased from MedChemExpress (MCE, New Jersey, USA).
3
Heterogeneous ACE2 Expression Construct
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The heterogeneous expression of ace2 cassette was constructed as follows. The primers PA2eno-1 and PA2eno-3, as well as TA2eno-4 and TA2eno-5 were used to amplify Peno and Teno from T. reesei QM9414 genomic DNA. Ace2 gene (GenBank accession no. CP016233.1) using primers ace2-F and ace2-R was also ampli ed from T. reesei QM9414 genomic DNA by PCR. The hygromycin resistance cassette was cloned from pUR5750 plasmid containing hygromycin resistance gene (hygromycin phosphotransferase, hph) which was preserved in our laboratory using primers PgpdA-hph-F and hph-TgtrpC-R. Then, the ace2 expression cassette could be obtained by fusion PCR with primers PA2eno-2 and TgtrpC-2 using the fragments mentioned above as the templates [22] . After that, primer hph-F and hph-R were used to verify the existence of the hygromycin resistance in the transformants, and then they were chosen after 96 h fermentation under the inducing condition of ABP medium. Sequences of all primers are given in Table1.
The DNA polymerases used in this study includes PrimeSTAR® HS DNA Polymerase (Takara, Japan) for amplifying 1-2kb gene segments, PrimeSTAR ® Max DNA Polymerase (Takara, Japan) for synthesizing 3kb-10kb gene segments, and the 2×Taq Plus Master Mix (Dye Plus) (Vazyme, China) for the PCR of verifying transformants.
The DNA polymerases used in this study includes PrimeSTAR® HS DNA Polymerase (Takara, Japan) for amplifying 1-2kb gene segments, PrimeSTAR ® Max DNA Polymerase (Takara, Japan) for synthesizing 3kb-10kb gene segments, and the 2×Taq Plus Master Mix (Dye Plus) (Vazyme, China) for the PCR of verifying transformants.
4
Brassinolide Regulation of Cotton
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For BL treatment, 10 µM/L BL to treat cotton seedlings growing the three-four leaf stage for 1, 3 and 5 hours. An equal volume of absolute ethanol (solvent) was added to deionized water as the Mock.
Samples of plant tissues were immediately stored in liquid nitrogen, and the extracted RNA was stored at -80℃. Each experiment was conducted with three biological replicates. We extracted total RNA using the RNA prep Pure Plant Kit (TSINGKE, Beijing, China) based on the manufacturer's instructions. EasyScript® (One-Step gDNA Removal) (TransGen Biotech, Beijing, China) was used to synthesize cDNA from 1 µg of RNA. The internal controls were Actin2 (AT3G18780.1) and GhHistone3 (AF024716). AceQ qPCR SYBR Green Master Mix (Low ROX Premixed) (Vazyme, Nanjing, China) was used for Quantitative Real-time PCR (qRT-PCR) analysis on a Light-Cycler 480 (Roche Diagnostics, Germany). Values of relative expression patterns were calculated using the 2 -△△Ct method (Livak and Schmittgen 2001) . 2⋅ Taq Plus Master Mix (Dye Plus) (Vazyme, Nanjing, China) was used for semi-quantitative RT-PCR analysis (Phillips et al. 1993 ).
Samples of plant tissues were immediately stored in liquid nitrogen, and the extracted RNA was stored at -80℃. Each experiment was conducted with three biological replicates. We extracted total RNA using the RNA prep Pure Plant Kit (TSINGKE, Beijing, China) based on the manufacturer's instructions. EasyScript® (One-Step gDNA Removal) (TransGen Biotech, Beijing, China) was used to synthesize cDNA from 1 µg of RNA. The internal controls were Actin2 (AT3G18780.1) and GhHistone3 (AF024716). AceQ qPCR SYBR Green Master Mix (Low ROX Premixed) (Vazyme, Nanjing, China) was used for Quantitative Real-time PCR (qRT-PCR) analysis on a Light-Cycler 480 (Roche Diagnostics, Germany). Values of relative expression patterns were calculated using the 2 -△△Ct method (Livak and Schmittgen 2001) . 2⋅ Taq Plus Master Mix (Dye Plus) (Vazyme, Nanjing, China) was used for semi-quantitative RT-PCR analysis (Phillips et al. 1993 ).
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