We determined sex by multiplexed polymerase chain reaction amplification of the two sex chromosome‐specific loci ZFX and SRY using the primers P1‐5EZ, P2‐3EZ (Aasen & Medrano, 1990 ) and Y53‐3C, Y53‐3D (Gilson et al., 1998 ), respectively. We used gel electrophoresis in combination with stained DNA bands (GelRedTM) and UV light (E‐Box; Vilber) to visually determine sex based on the number of visible bands.
E box
Manufactured by Vilber
Sourced in France
The E-Box is a laboratory equipment designed for the visualization and documentation of electrophoresis gels. It features a high-resolution camera and LED illumination to capture clear images of gel samples.
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5 protocols using e box
1
Multiplexed PCR Sex Determination
2
Dichelobacter nodosus Serogrouping by PCR
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Dichelobacter nodosus serogrouping was performed using the multiplex PCR of nine (A–I) serogroup-specific primers as described previously by Dhungyel et al. [10 (link)]. The PCR products were analyzed in 2% agarose gels and visualized under ultraviolet illumination using an E-Box Vilber (Vilber, France).
3
Quantifying HAC1 mRNA Splicing Efficiency
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Total RNA samples, which were extracted from yeast cells using the hot-phenol method (Collart and Oliviero, 2001 (link)), were subjected to RT-PCR in which the poly(dT) RT primers and the HAC1-specific PCR primers were employed (Promlek et al., 2011 (link); Mai et al., 2018 (link)). As the forward and reverse PCR primers interposed the HAC1-intron sequence, RT-PCR yielded different-sized products from unspliced (HAC1u) and spliced (HAC1i) HAC1 mRNAs. The RT-PCR products were electrophoresed on a 2% agarose gel, and the ethidium bromide-fluorescent image was captured with a UV-transilluminating imager E-Box (Vilber Lourmat). The gel images were analyzed using ImageJ software (https://imagej.nih.gov/ij/ ), and the HAC1 mRNA-splicing efficiency was calculated using the following formula:
To check the XBP1 mRNA splicing pattern in HeLa cells, we extracted total RNA and performed RT-PCR analysis as described by Tsuchiya et al. (2018) (link). The sequences of the human XBP1 specific PCR primers were 5′-TTACGAGAGAAAACTCATGGCC-3′ and 5′-GGGTCCAAGTTGTCCAGAATGC-3′.
To check the XBP1 mRNA splicing pattern in HeLa cells, we extracted total RNA and performed RT-PCR analysis as described by Tsuchiya et al. (2018) (link). The sequences of the human XBP1 specific PCR primers were 5′-TTACGAGAGAAAACTCATGGCC-3′ and 5′-GGGTCCAAGTTGTCCAGAATGC-3′.
4
Characterization of Small Molecule Ligands
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AVANCE III 400 and 300 NMR spectrometers (Bruker Corporation, Billerica, MA, USA) were used to record the 1H-NMR spectra of the small molecule ligands, and CDCl3 was used as the solvent.
A MALDI-TOF Autoflex Speed mass spectrometer (Bruker Corporation, Billerica, MA, USA) or an Agilent G6410A LC-MS/MS Instrument (Agilent Technologies, Santa Clara, CA, USA) was used for the recording of mass spectra.
A NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the oligonucleotide solutions’ optical densities.
After analytical gel-electrophoresis, the gels were either stained with a Stains-all dye for qualitative visualization or stained with ethidium bromide and quantified using the E-Box (Vilber, Marne-la-Vallée, France).
A MALDI-TOF Autoflex Speed mass spectrometer (Bruker Corporation, Billerica, MA, USA) or an Agilent G6410A LC-MS/MS Instrument (Agilent Technologies, Santa Clara, CA, USA) was used for the recording of mass spectra.
A NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the oligonucleotide solutions’ optical densities.
After analytical gel-electrophoresis, the gels were either stained with a Stains-all dye for qualitative visualization or stained with ethidium bromide and quantified using the E-Box (Vilber, Marne-la-Vallée, France).
5
Evaluating Nucleic Acid Protection by Vectors
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Vectors in a concentration of 0.03 μg DNA/μL were subjected to electrophoresis on an ethidium bromide-containing gel (1% agarose). Subsequently, bands were photographed with a Vilber (Vilber Lourmat, Marne-la-Vallée, France) E-BOX. In the protection study, 1 U of DNase and 1.2 μg DNA (Sigma-Aldrich) were incubated (37°C) with the vectors and complexes for 30 minutes. Then, a 2% sodium dodecyl sulfate (SDS) solution was added as a DNA release reagent. Samples were subjected to agarose gel electrophoresis and compared to untreated DNA.
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