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Dithiothreitol (dtt)

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Dithiothreitol (DTT) is a reducing agent commonly used in biochemical applications. It acts as a disulfide bond reducer, facilitating the maintenance of proteins in their reduced state.

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Lab products found in correlation

Nextseq 500 sequencer, by Illumina (2 mentions) Rnase inhibitor, by Promega (2 mentions) Dntp mix, by New England Biolabs (2 mentions) Rnasin plus, by Promega (2 mentions) Smartscribe, by Takara Bio (2 mentions) Smart seq2 lysis buffer, by New England Biolabs (2 mentions) Oligo dt30vn primer, by New England Biolabs (2 mentions) Triton x 100 10, by Merck Group (2 mentions) Smart first strand buffer, by Takara Bio (2 mentions) Rnase inhibitor, by Takara Bio (2 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Oligo dt, by Promega (1 mentions) Sw40ti rotor, by Beckman Coulter (1 mentions) Syto9, by Thermo Fisher Scientific (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) Ampicillin, by Merck Group (1 mentions) Kanamycin, by Merck Group (1 mentions) Syto 9, by Takara Bio (1 mentions) Mncl2 4h2o, by Merck Group (1 mentions) Amphotericin b, by Merck Group (1 mentions)

6 protocols using dithiothreitol (dtt)

1

Single-cell transcriptomics of bone marrow

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Lineage-depleted bone marrow cells were obtained by crushing and stained with the following antibodies on ice for 30 min: CD41, CD45, CD51, CD61, CD71, CD200, Ter119 and VCAM1 (Table S4). Indexed single-cells were sorted into 5μl of Smart-Seq2 lysis buffer (2μM oligo-dT30VN primer, 2mM dNTP mix (10mM each, NEB), 1:50 RNAse inhibitor (promega) and 1:125 Triton X-100 10% (Sigma-Aldrich)) and immediately snap frozen in an ethanol and dry ice bath. Plates were kept at -80 °C until processing. cDNA amplification was performed using a modified Smart-Seq2 protocol by adding, after 3 min at 72 °C, 5μl of RT mix containing 1× SMART First Strand Buffer (Clontech), 2 mM dithiothreitol (Clontech), 2 μM template switching oligo (Exiqon), 10 U μl-1 SMARTScribe (Clontech) and 10 U μl-1 RNASin plus (Promega). Transcriptome amplification was performed using 1X KAPA HiFi HS MM and 0.1μM ISPCR primer, with 21 PCR enrichment cycles. Libraries were constructed using in house produced Tn554 (link) at 1:100 dilution and sequenced on an Illumina Next-Seq 500 sequencer, with 75 cycles single end sequencing.
Baccin C., Al-Sabah J., Velten L., Helbling P.M., Grünschläger F., Hernández-Malmierca P., Nombela-Arrieta C., Steinmetz L.M., Trumpp A, & Haas S. (2019). Combined single-cell and spatial transcriptomics reveals the molecular, cellular and spatial bone marrow niche organization. Nature cell biology, 22(1), 38-48.
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2

Single-cell transcriptomics of bone marrow

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Lineage-depleted bone marrow cells were obtained by crushing and stained with the following antibodies on ice for 30 min: CD41, CD45, CD51, CD61, CD71, CD200, Ter119 and VCAM1 (Table S4). Indexed single-cells were sorted into 5μl of Smart-Seq2 lysis buffer (2μM oligo-dT30VN primer, 2mM dNTP mix (10mM each, NEB), 1:50 RNAse inhibitor (promega) and 1:125 Triton X-100 10% (Sigma-Aldrich)) and immediately snap frozen in an ethanol and dry ice bath. Plates were kept at -80 °C until processing. cDNA amplification was performed using a modified Smart-Seq2 protocol by adding, after 3 min at 72 °C, 5μl of RT mix containing 1× SMART First Strand Buffer (Clontech), 2 mM dithiothreitol (Clontech), 2 μM template switching oligo (Exiqon), 10 U μl-1 SMARTScribe (Clontech) and 10 U μl-1 RNASin plus (Promega). Transcriptome amplification was performed using 1X KAPA HiFi HS MM and 0.1μM ISPCR primer, with 21 PCR enrichment cycles. Libraries were constructed using in house produced Tn554 (link) at 1:100 dilution and sequenced on an Illumina Next-Seq 500 sequencer, with 75 cycles single end sequencing.
Baccin C., Al-Sabah J., Velten L., Helbling P.M., Grünschläger F., Hernández-Malmierca P., Nombela-Arrieta C., Steinmetz L.M., Trumpp A, & Haas S. (2019). Combined single-cell and spatial transcriptomics reveals the molecular, cellular and spatial bone marrow niche organization. Nature cell biology, 22(1), 38-48.
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3

RT-PCR Detection of mRNA Transcripts

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Detection of mRNA transcripts was performed as previously described (27 (link)). Briefly, after culture in growth medium for 2 days, TC1, AKR and AKR-E7 cells were harvested and homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA). Three μg of RNA was reverse transcribed using 0.5 μg oligo (dT) (Promega, Madison, WI), 1 mmol/L deoxynucleotide triphosphates, 10 mmol/L dithiothreitol (Clontech), and 10 units SuperScript III reverse transcriptase in 1× First-Strand Buffer (Clontech, Palo Alto, CA) with 20 μl final volume for 60 minutes at 50°C, 15 minutes at 70°C and 5 minutes at 99°C. Primers were obtained from the literature or designed using standard protocols. Primer sequences can be obtained from the authors on request. cDNA concentrations from each pool were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control gene. PCR products were electrophoresed in a 1.5% agarose gel containing ethidium bromide.
Quatromoni J.G., Predina J.D., Bhojnagarwala P., Judy R.P., Jiang J., De Jesus E.M., Kapoor V., Cheng G., Okusanya O.T., Eruslanov E, & Singhal S. (2014). Adenoviral Based Immunotherapy Provides Local Disease Control in an Orthotopic Murine Model of Esophageal Cancer. Journal of immunotherapy (Hagerstown, Md. : 1997), 37(5), 283-292.
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4

Ribosome Profiling of Tau-Knockout Mice

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Cerebral cortices and hippocampi from 34-week old wild-type and tau-knockout CB57BL/6J mice were prepared by the Institute of Immunology, Utsunomiya (Japan). Briefly, brains were washed with ice-cold PBS and homogenized in TKM buffer (50 mM triethanolamine [pH 7.8], 50 mM KCl, 5 mM MgCl2, 0.25 M sucrose, 1 mM PMSF, protein inhibitor [complete cocktail without EDTA, Roche], 1 mM DTT and RNase inhibitor [0.2 unit/μl, Takara]). Homogenates were centrifuged (1000 × g, 10 min, 4 °C) and 0.5 ml of each supernatant was loaded onto a 15–45% sucrose gradient (9 ml) with a 0.75 ml cushion (45% sucrose) before centrifugation at 36,000 rpm (2 h, 4 °C, Beckman Coulter SW40Ti rotor). The gradient was fractionated, and the distribution of RNAs and ribosomal protein in each fraction was monitored by absorbance at 254 nm and Western blotting of S6 ribosome protein, respectively.
Kobayashi S., Tanaka T., Soeda Y., Almeida O.F, & Takashima A. (2017). Local Somatodendritic Translation and Hyperphosphorylation of Tau Protein Triggered by AMPA and NMDA Receptor Stimulation. EBioMedicine, 20, 120-126.
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5

Fluorescence-based SARS-CoV-2 RdRp Assay

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The fluorescence emitted was recorded by employing GloMax Discover (Promega, USA) using the excitation and emission filters at 485 and 520 nm. This assay records the synthesis of dsRNA in a reaction using ATP (Sigma Aldrich) as a nucleotide substrate and a poly-U (Sigma Aldrich) molecule as a template using fluorescent dye SYTO 9 (Invitrogen), which binds only to dsRNA [24 (link)]. The standard reaction contained 50 mM Tris-HCl (pH 8.0), 5 mM MnCl2·4H2O (Sigma Aldrich), 50 mM NaCl (Samchun), 4 mM DTT (TaKaRa), 3 mM ATP, 13 μg/ml poly-U, and 0.25 μM SYTO 9. The assay was initiated by adding 1 μg/μl RdRp/NSP7/NSP8 (SARS-CoV-2) Complex (BPS Bioscience, USA), and the fluorescence was recorded over 20 min at 30°C. The reaction was conducted in black, 96-well, flat-bottomed plates. For the compound used in this assay, NPP B1 with a purity of over 80% on HPLC was used, and ampicillin, kanamycin, and amphotericin B were purchased from Sigma Aldrich.
Park J.H., Park H.S., Nah H.J., Kang S.H., Choi S.S, & Kim E.S. (2022). Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor. Journal of Microbiology and Biotechnology, 32(7), 911-917.
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6

5' RACE for cDNA Synthesis

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5′ Rapid amplification of cDNA ends (RACE) was used to generate cDNA from RNA using the Takara Bio SMARTer reverse transcriptase kit. Master mix was made up of 2 μl 5x First Strand Buffer (Takara Bio), 1 μl 20 mM DTT, 1 μl 10 mM dNTPs (Takara), 1 μl 12 μM SMARTer IIA oligo, 0.5 μl 20 U/μl RNase Inhibitor (Takara) and 1 μl 100 U/μl SMARTScribe Reverse Transcriptase. 1 μl 5′-CDS Primer A was then added to 2.75 μl of each sample RNA and incubated at 72 °C for 3 min, then cooled to 4 °C for 2 min. The RNA and CDS primer was then added to the master mix and incubated at 42 °C for 90 mins then 70 °C for 10 mins in a Thermocycler. 20 μl Tris-EDTA was then added to the resulting mix and the samples stored at − 20 °C.
Dixon R., Preston S.G., Dascalu S., Flammer P.G., Fiddaman S.R., McLoughlin K., Boyd A., Volf J., Rychlik I., Bonsall M.B., Kaspers B, & Smith A.L. (2021). Repertoire analysis of γδ T cells in the chicken enables functional annotation of the genomic region revealing highly variable pan-tissue TCR gamma V gene usage as well as identifying public and private repertoires. BMC Genomics, 22, 719.
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