Second strand synthesis enzyme mix

Manufactured by New England Biolabs

The Second Strand Synthesis Enzyme mix is a reagent used in molecular biology protocols to synthesize the second strand of complementary DNA (cDNA) from a single-stranded RNA template. The mix contains the necessary enzymes and cofactors to efficiently catalyze this reaction.

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Lab products found in correlation

G 50 columns, by GE Healthcare (4 mentions) Qubit dsdna hs kit, by Thermo Fisher Scientific (1 mentions) Nebnext hairpin adaptors, by New England Biolabs (1 mentions) High sensitivity dna kit for bioanalyzer, by Agilent Technologies (1 mentions) Hiseq 2500, by Illumina (1 mentions) Ampure xp bead, by Beckman Coulter (1 mentions) Second strand synthesis reaction buffer, by New England Biolabs (1 mentions) Nextera xt library prep kit, by Illumina (1 mentions) Illustra microspin g 50 column, by GE Healthcare (1 mentions) Agencourt ampure xp bead, by Beckman Coulter (1 mentions)

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NEBNext Second Strand Synthesis Enzyme Mix (4 citations)

6 protocols using second strand synthesis enzyme mix

RNA-seq Library Preparation and Sequencing

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RNA-seq libraries were constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) following the manufacturer’s instructions. Briefly, mRNA isolated from ∼1 μg of total RNA was fragmented to an average size of 200 nt by incubating at 94° for 15 min in first strand buffer, cDNA was synthesized using random primers and ProtoScript II Reverse Transcriptase followed by second strand synthesis using NEB Second Strand Synthesis Enzyme Mix. Resulting DNA fragments were end-repaired, dA tailed and ligated to NEBNext hairpin adaptors (NEB #E7335). After ligation, adaptors were converted to the ‘Y’ shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter #A63880) to generate fragment sizes between 250 and 350 bp. Adaptor-ligated DNA was PCR amplified followed by AMPure XP bead clean up. Libraries were quantified with Qubit dsDNA HS Kit (ThermoFisher Scientific #Q32854) and the size distribution was confirmed with High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies #5067- 4626). Libraries were sequenced on Illumina HiSeq2500 in single read mode, with an approximately similar depth of 30 million reads per sample, and a read length of 50 nt following manufacturer’s instructions. Base calls were performed with RTA 1.18.64 followed by conversion to FASTQ with bcl2fastq 1.8.4.

Gamez S., Antoshechkin I., Mendez-Sanchez S.C, & Akbari O.S. (2020). The Developmental Transcriptome of Aedes albopictus, a Major Worldwide Human Disease Vector. G3: Genes|Genomes|Genetics, 10(3), 1051-1062.

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Sequencing-Based RNA Structure Analysis

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From each sample of RNA (defined above, E. coli or murine total RNA, extracted and SHAPEmodified or untreated), 1-3 μg were subjected to MaP reverse transcription [requiring Superscript II and addition of Mn²⁺ to the RT buffer(25 (link), 39 (link))] using random nonamer primers. The cDNA generated was buffer exchanged (Illustra microspin G-50 columns, GE Healthcare) and the volume increased to 68 μL. For second-strand cDNA synthesis, 8 μL of 10× buffer (Second Strand Synthesis Reaction Buffer, NEB) and 4 μL enzyme (Second Strand Synthesis Enzyme mix, NEB) were added to the cDNA product and incubated for 2.5 h at 16 °C. Double-stranded cDNA was fragmented and amplified with sequencing indexes (Nextera XT library prep kit, Illumina). Nextera PCR products were affinity purified (using a 0.8× ratio of Agencourt AMPure XP beads, Beckman Coulter) and eluted in 20 μL of nuclease-free water.

Busan S., Weidmann C.A., Sengupta A, & Weeks K.M. (2019). Guidelines for SHAPE reagent choice and detection strategy for RNA structure probing studies. Biochemistry, 58(23), 2655-2664.

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Mutational Profiling of pri-miR-17~92 RNA

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After pri-miR-17∼92 RNA was dephosphorylated, mutational profiling reverse transcription (RT) was performed. 1 μl of nonamer primer [200 ng/μl or 2μM] was added to 1-3 μg of RNA in 10 μl nuclease free water. The samples were incubated at 65°C for 5 min then cooled on ice. 8 μl of [2.5x] MaP buffer (125 mM Tris pH 8.0, 187.5 mM KCl, 15 mM MnCl₂, 25 mM DTT, and 1.25 mM dNTPs) was added and incubated at 42°C for 2 min. 1 μl of SuperScript II reverse transcriptase was added and mixed well before incubating the reaction at 42°C for 2-3 h, and then at 70°C to inactivate the polymerase. cDNA was exchanged into water using G-50 columns (GE Life Sciences) the volume increased to 68 μl using nuclease free water. Second strand synthesis was carried out (Second Strand Synthesis Enzyme mix; New England Biolabs) and the dsDNA was used to generate a Nextera library for sequencing on an Illumina MiSeq, as described.⁵² (link)

Stephenson W., Razaghi R., Busan S., Weeks K.M., Timp W, & Smibert P. (2022). Direct detection of RNA modifications and structure using single-molecule nanopore sequencing. Cell Genomics, 2(2), 100097.

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RNA Structure Probing with SHAPE-MaP

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Extracted rRNA was treated with NAI [100 mM final], AcIm [100 mM final], NMIA [13 mM final] or DMSO (unmodified control). All SHAPE-MaP experiments were performed with 10% volume fraction of DMSO. Modification was carried out at 37°C for 3 half-lives of the chemical probe used. For mutational profiling RT, 1 μl of nonamer primer [200 ng/μl or 2μM] was added to 1-3 μg of rRNA in 10 μl nuclease free water. The samples were incubated at 65°C for 5 min then cooled on ice. 8 μl of [2.5x] MaP buffer (125 mM Tris pH 8.0, 187.5 mM KCl, 15 mM MnCl₂, 25 mM DTT, and 1.25 mM dNTPs) was added and incubated at 42°C for 2 min. 1 μl of SuperScript II reverse transcriptase was added and mixed well before incubating the reaction at 42°C for 2-3 h, and then at 70°C to inactivate the polymerase. cDNA was exchanged into water using G-50 columns (GE Life Sciences) the volume increased to 68 μl using nuclease free water. Second strand synthesis was carried out (Second Strand Synthesis Enzyme mix; New England Biolabs) and the dsDNA was used to generate a Nextera library for sequencing on an Illumina MiSeq, as described.⁵² (link)

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Mutational Profiling of Structured RNA

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Extracted rRNA was treated with NAI [100 mM final], AcIm [100 mM final], NMIA [13 mM final] or DMSO (unmodified control). All SHAPE-MaP experiments were performed with 10% volume fraction of DMSO. Modification was carried out at 37°C for 3 half-lives of the chemical probe used. For mutational profiling RT, 1 μl of nonamer primer [200 ng/μl or 2μM] was added to 1–3 μg of rRNA in 10 μl nuclease free water. The samples were incubated at 65°C for 5 min then cooled on ice. 8 μl of [2.5x] MaP buffer (125 mM Tris pH 8.0, 187.5 mM KCl, 15 mM MnCl₂, 25 mM DTT, and 1.25 mM dNTPs) was added and incubated at 42°C for 2 min. 1 μl of SuperScript II reverse transcriptase was added and mixed well before incubating the reaction at 42°C for 2–3 h, and then at 70°C to inactivate the polymerase. cDNA was exchanged into water using G-50 columns (GE Life Sciences) the volume increased to 68 μl using nuclease free water. Second strand synthesis was carried out (Second Strand Synthesis Enzyme mix; New England Biolabs) and the dsDNA was used to generate a Nextera library for sequencing on an Illumina MiSeq, as described.^{52 (link)}

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Mutational Profiling of pri-miR-17~92

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After pri-miR-17~92 RNA was dephosphorylated, mutational profiling reverse transcription (RT) was performed. 1 μl of nonamer primer [200 ng/μl or 2μM] was added to 1–3 μg of RNA in 10 μl nuclease free water. The samples were incubated at 65°C for 5 min then cooled on ice. 8 μl of [2.5x] MaP buffer (125 mM Tris pH 8.0, 187.5 mM KCl, 15 mM MnCl₂, 25 mM DTT, and 1.25 mM dNTPs) was added and incubated at 42°C for 2 min. 1 μl of SuperScript II reverse transcriptase was added and mixed well before incubating the reaction at 42°C for 2–3 h, and then at 70°C to inactivate the polymerase. cDNA was exchanged into water using G-50 columns (GE Life Sciences) the volume increased to 68 μl using nuclease free water. Second strand synthesis was carried out (Second Strand Synthesis Enzyme mix; New England Biolabs) and the dsDNA was used to generate a Nextera library for sequencing on an Illumina MiSeq, as described.^{52 (link)}

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