Superscript 3 rt enzyme

Manufactured by Thermo Fisher Scientific

Sourced in United States

Superscript III RT enzyme is a reverse transcriptase enzyme used for the synthesis of complementary DNA (cDNA) from RNA templates. It is a thermostable enzyme derived from Moloney Murine Leukemia Virus (MMLV) and is optimized for high-sensitivity, high-yield reverse transcription reactions.

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33 protocols using superscript 3 rt enzyme

Quantitative RT-PCR Analysis of Liver Tissue

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Liver tissue was mechanically homogenized in Trizol (Life Technologies, Carlsbad, CA, USA) using the manufacturer’s method, followed by purification using Direct-zol RNA miniPrep (ZymoResearch, Irvine, CA, USA) with on column DNAse 1 digestion. Quality was analysed on agarose gels. cDNA synthesis was performed with 1 μg total sample RNA, using random hexamers and Superscript III RT enzyme (Life Technologies). Quantitative real-time PCR was carried out as described earlier using iCycler iQ (Bio-Rad, Hercules, CA, USA) and SYBR green fluorescence to detect double-stranded DNA. Values were normalized to controls, ACTB, TATABP and PPIA. Results are reported as mean values. There were 5 and 7 mice in the DEN injected groups and 3 each in the two mock injected groups. All PCR runs included reference cDNA to allow comparison of expression levels of samples tested at different times. Primer sets used are described in the supplementary material.

Baranski O.A., Kalinichenko V.V, & Adami G.R. (2014). Increased FOXM1 expression can stimulate DNA repair in normal hepatocytes in vivo but also increases nuclear foci associated with senescence. Cell Proliferation, 48(1), 105-115.

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RNA-Seq Analysis of JW23.3 Cell Responses

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JW23.3 cells were treated with 40 μM WU-12a, 40 μM WU-76, or vehicle control (DMSO) for 48 hours. RNA was isolated as described above, and samples contained at least 5 ug of purified total RNA with RIN > 9.0. At least three biological replicates were performed. Samples were aligned against Mouse Ensembl GRCm38.76. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. Differential expression analysis was performed with the DESeq2 package (R Bioconductor software; RRID:SCR_015687).

Borcherding D.C., Amin N.V., He K., Zhang X., Lyu Y., Dehner C., Bhatia H., Gothra A., Daud L., Ruminski P., Pratilas C.A., Pollard K., Sundby T., Widemann B.C, & Hirbe A.C. (2023). MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors. Clinical Cancer Research, 29(8), 1592-1604.

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RNA-seq Library Preparation and Sequencing

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Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 500 ng to 1 μg of total RNA. Ribosomal RNA was blocked using FastSelect reagents (Qiagen) during complementary DNA synthesis. RNA was fragmented in reverse transcriptase buffer with FastSelect reagent and heated to 94 °C for 5 min, 75 °C for 2 min, 70 °C for 2 min, 65 °C for 2 min, 60 °C for 2 min, 55 °C for 2 min, 37 °C for 5 min and 25 °C for 5 min. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield double-stranded cDNA (ds-cDNA). cDNA was blunt ended, had an A base added to the 3′ ends and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 S4 instrument, generating approximately 30 million paired-end 2 × 150 reads per library.

Cui Zhou D., Jayasinghe R.G., Chen S., Herndon J.M., Iglesia M.D., Navale P., Wendl M.C., Caravan W., Sato K., Storrs E., Mo C.K., Liu J., Southard-Smith A.N., Wu Y., Naser Al Deen N., Baer J.M., Fulton R.S., Wyczalkowski M.A., Liu R., Fronick C.C., Fulton L.A., Shinkle A., Thammavong L., Zhu H., Sun H., Wang L.B., Li Y., Zuo C., McMichael J.F., Davies S.R., Appelbaum E.L., Robbins K.J., Chasnoff S.E., Yang X., Reeb A.N., Oh C., Serasanambati M., Lal P., Varghese R., Mashl J.R., Ponce J., Terekhanova N.V., Yao L., Wang F., Chen L., Schnaubelt M., Lu R.J., Schwarz J.K., Puram S.V., Kim A.H., Song S.K., Shoghi K.I., Lau K.S., Ju T., Chen K., Chatterjee D., Hawkins W.G., Zhang H., Achilefu S., Chheda M.G., Oh S.T., Gillanders W.E., Chen F., DeNardo D.G., Fields R.C, & Ding L. (2022). Spatially restricted drivers and transitional cell populations cooperate with the microenvironment in untreated and chemo-resistant pancreatic cancer. Nature Genetics, 54(9), 1390-1405.

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Transcriptomic Analysis of Dorsal Root Ganglia

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At 12, 24 or 72 h after nerve lesion, L4-5 DRG tissues were dissected for RNA isolation and the tail tissues were collected for confirmative genotyping experiment. From the mice in the 12 h group, the contralateral DRGs were also collected as uninjured (u) control. Total RNA was isolated from the DRG tissues with RNAqueous-Micro Total RNA Isolation Kit (ThermoFisher, AM1931). Then, RNA from two individual mice were pooled per sample and subjected to DNase I reaction by using RNA Clean & Concentrator kit (Zymo research). RNA integrity was determined using an Agilent Bioanalyzer and ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina). mRNA was then fragmented in buffer containing 40mM Tris Acetate pH 8.2, 100mM Potassium Acetate and 30mM Magnesium Acetate and heating to 94 degrees for 150 seconds. mRNA was reverse transcribed to yield cDNA using Superscript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 14 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-2500 using single reads extending 50 bases targeting 30 million reads per sample.

Shin J.E., Ha H., Kim Y.K., Cho Y, & DiAntonio A. (2019). DLK regulates a distinctive transcriptional regeneration program after peripheral nerve injury. Neurobiology of disease, 127, 178-192.

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Illumina-Based RNA Sequencing Protocol

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RNA sequencing was performed by the Washington University Genome Technology Access Center) as we reported^{51 (link)}. Library preparation was performed with 10 μG of total RNA with a Bioanalyzer RIN score >8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was fragmented and reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies) and random hexamers. A second strand reaction was performed to yield ds-cDNA, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were amplified for 12 cycles and sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases. RNA-seq reads were aligned to the Ensembl release 76 top-level assembly with STAR version 2.0.4b. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. Transcript counts were produced by Sailfish version 0.6.3.

Higgins C.B., Mayer A.L., Zhang Y., Franczyk M., Ballentine S., Yoshino J, & DeBosch B.J. (2022). SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase. Nature Communications, 13, 1074.

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RNA Sequencing with Illumina NovaSeq

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For RNA sequencing, we first removed ribosomal RNA by an RNase-H method using RiboErase kits (Kapa Biosystems). Then, we fragmented mRNA and then reverse transcribed mRNA to cDNA using SuperScript III RT enzyme (Life Technologies) and random hexamers following the manufacturer’s instructions. We performed a second strand reaction to yield ds-cDNA. Then, we blunt-ended the cDNA, added an A base to the 3′ ends, and then ligated Illumina sequencing adapters to the ends. We amplified ligated fragments and incorporated unique dual index tags. We sequenced fragments on an Illumina NovaSeq 6000 using paired end reads extending 150 bases at the McDonnell Genome Institute at Washington University School of Medicine in St. Louis. RNA sequencing reads were then aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3-8 software. We identified 1 of the 8 control samples to be an outlier and excluded it from further analyses.

Lin J.B., Santeford A., Colasanti J.J., Lee Y., Shah A.V., Wang T.J., Ruzycki P.A, & Apte R.S. (2024). Targeting cell-type-specific, choroid-peripheral immune signaling to treat age-related macular degeneration. Cell Reports Medicine, 5(1), 101353.

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Transcriptional Profiling of Azadirachta indica

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Total RNA from leaf, callus and developing endosperm (S1 =10 days post seed setting, S2 = 20 days post seed setting, S3 = 30 days post seed setting, S4 = 40 days post seed setting) was isolated using TRIzol reagent method and quantified using a Qubit Fluorometer. The cDNA synthesis was performed using total RNA (1 µg) with oligo(dT) random primers (50 µM) and SuperScript^® III RT enzyme (200 u/µl) (Cat # 18080044; Life Technologies, Carlsbad, California, USA). The qPCR was performed on an Applied Biosystems, 7900HT Fast Real-Time PCR system machine. Real-time PCR was performed in a 384-wells optical reaction plate (Applied Biosystems, Foster City, California, USA) using SYBR green PCR mastermix (Life Technologies, Cat #4344463), which contains AmpliTaq Gold^® DNA polymerase and ROX as a passive reference dye. Cycling conditions were 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s with 40 cycles. We compared the fold change in qPCR experiments for selected genes from azadirachtin biosynthesis with conserved eukaryotic (rice) elongation factor 1α (eEF-1α) gene (AK061464.1). All reactions were performed in triplicate with elongation factor primers and water as an internal control. The primer sequences of selected genes are listed in the Table S6.

Kuravadi N.A., Yenagi V., Rangiah K., Mahesh H., Rajamani A., Shirke M.D., Russiachand H., Loganathan R.M., Shankara Lingu C., Siddappa S., Ramamurthy A., Sathyanarayana B, & Gowda M. (2015). Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree. PeerJ, 3, e1066.

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RNA-Seq of C4-2B cells treated with (R)-9b

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C4-2B cells (5 × 10⁷ cells) were either treated with vehicle or (R)-9b. Total RNA was extracted using RNeasy kit from QIAGEN (Cat #74136). Total RNA integrity was determined using Agilent Bioanalyzer or 4200 TapeStation. Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 min. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12–15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. Base calls and demultiplexing were performed with Illumina’s bcl2fastq software with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 101 primary assembly with STAR version 2.7.9a1. Gene counts were derived from the number of uniquely aligned unambiguous reads.

Sridaran D., Chouhan S., Mahajan K., Renganathan A., Weimholt C., Bhagwat S., Reimers M., Kim E.H., Thakur M.K., Saeed M.A., Pachynski R.K., Seeliger M.A., Miller W.T., Feng F.Y, & Mahajan N.P. (2022). Inhibiting ACK1-mediated phosphorylation of C-terminal Src kinase counteracts prostate cancer immune checkpoint blockade resistance. Nature Communications, 13, 6929.

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Quantifying Gene Expression in Mouse Models

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RNA was isolated using RNAeasy kit (Qiagen) from ST HDH Q7/111 cells and striatal tissue of mice injected with Kv4.2 shRNA or ZFP AAV. The RNA was reverse transcribed with Superscript III RT enzyme (Life Technologies) or qScript cDNA synthesis kit (Quanta Biosciences). Quantitative real-time PCR was performed using an ABI StepOnePlus Real Time PCR system with SYBR-Green PCR Master Mix (Applied Biosystems, Forster City, CA). The relative abundance of different transcripts was assessed by SYBR quantitative PCR in triplicate.
The following primers (Integrated DNA Technologies) were used for PCR amplification: GAPDH (accession number: NM_008084.2) mGAPDH-F ′-CATTTGCAGTGGCAAAGTGG-3′, mGAPDH-R 5′-GAATTTGCCGTGAGTGGAGT-3′; Kv4.2-F 5'- GTGTCGGGAAGCCATAGAGGC-3', Kv4.2-R 5'- TTACAAGGCAGACACCCTGA-3; wild-type-mouse Htt_fw: CAG GTC CGG CAG AGG AAC C, Mut-mouse-Htt_Q175_fw: GCC CGG CTG TGG CTG A, Mut and wild-type Htt_rv*: TTC ACA CGG TCT TTC TTG GTG G (*wild-type and mutant Htt share the same reverse primer sequence (Htt gene accession number: NM_010414.3)). Experimental Ct values were normalized to GAPDH values using the formula: ΔCt = Ct (Kv4.2 or Htt) - Ct (GAPDH). The final expression levels were shown as ΔCt values. The PCR products were verified by melt curve analysis and agarose gels.

Carrillo-Reid L., Day M., Xie Z., Melendez A.E., Kondapalli J., Plotkin J.L., Wokosin D.L., Chen Y., Kress G.J., Kaplitt M., Ilijic E., Guzman J.N., Chan C.S, & Surmeier D.J. (2019). Mutant huntingtin enhances activation of dendritic Kv4 K+ channels in striatal spiny projection neurons. eLife, 8, e40818.

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Transcriptional Profiling of JW23.3 Cells

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JW23.3 cells were treated with 40 μmol/L WU-12a, 40 μmol/L WU-76, or vehicle control (DMSO) for 48 hours. RNA was isolated as described above, and samples contained at least 5 μg of purified total RNA with RIN >9.0. At least three biological replicates were performed. Samples were aligned against Mouse Ensembl GRCm38.76. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 5 to 10 μg of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct Kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94° for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 to 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. Differential expression analysis was performed with the DESeq2 package (R Bioconductor software; RRID:SCR_015687).

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