Product Class: Other

Template Switching RT Enzyme Mix 
cloned at NEB recombinant 80 Heat hotstart

Product Introduction

The Template Switching RT Enzyme Mix and accompanying reaction buffer enable efficient template switching activity in a reverse transcription reaction. The mix contains a unique RT and Murine RNase Inhibitor. Unlike competitor RT products, no additives (such as PEG or betaine) are required for optimal performance, simplifying reaction setup. In conjunction with a template switching oligo (TSO), cDNA is synthesized with a known sequence of choice attached to the 3′ end. The resulting cDNA can be amplified by PCR or serve as template for 5′ RACE (rapid amplification of cDNA ends) or 2nd strand cDNA synthesis.

  • Prepare RNA-seq libraries from extremely low input: single cells/nuclei or 2 pg of total RNA.
  • Perform 5′ RACE from as low as 10 ng total RNA.
  • Low background for RNA-seq or 5′ RACE
  • Enzyme mix contains Murine RNase Inhibitor, no additives necessary
  • Compatible with various template switching oligos (TSOs), RT primers, and DNA polymerases for full length cDNA amplification
  • Enjoy faster protocols as compared to alternative RNA-seq methods (eg. Smart-Seq®)
Catalog # Size Concentration
M0466S 20.0 reactions 10 X
M0466L 100.0 reactions 10 X

Product Information

Description

The reverse transcriptase (RT) in the Template Switching RT Enzyme Mix adds a few non-templated nucleotides after it reaches the 5′ end of the RNA template. These non-templated nucleotides can anneal to a template switching oligo (TSO) with a known sequence, prompting the reverse transcriptase to switch template from RNA to the TSO. The resulting cDNA contains a known sequence (complementary to the sequence of the TSO) attached to the 3′ end. This feature can be utilized in a variety of downstream applications, such as cDNA amplification, 5´ RACE (rapid amplification of cDNA ends), and 2nd strand cDNA synthesis. Optimized protocols are located in the Protocols section.
 
Figure 1: Template Switching Overview



Upon reaching the 5′ end of the RNA template, the reverse transcriptase adds a few non-templated nucleotides to the 3′ end of the cDNA. These non-templated nucleotides can anneal to a template switching oligo with a known sequence handle of choice, prompting the reverse transcriptase to switch from the RNA template to the TSO. The resulting cDNA contains a universal sequence (complementary to the TSO sequence) at the 3' end.
 


Figure 2: Template Switching RT Enzyme Mix performance advantages to Smart-seq2 method for single cell RNA-seq



A. Overview of template switching-mediated cDNA amplification. The RT primer contains a 5´ adaptor which, in conjunction with TSO, adds adaptors to both 5′ and 3′ ends of the cDNA. The entire RT reaction is subsequently PCR amplified with primers recognizing the adaptor sequences.
 
B-D. cDNA libraries were generated from 10 pg of Universal Human Reference (UHR) RNA (Agilent®) with ERCC RNA Spike-In Mix I (Thermo Fisher Scientific®) using NEB Template Switching RT Enzyme Mix or the Smart-seq2 method as described in Picelli, S. et al. (2014). Nat. Protoc. 9, 171-81. Each cDNA library was made into Illumina libraries using the NEBNext® Ultra™ II FS DNA Library Prep Kit (NEB #E7805) and sequenced using 2x75 cycles on a Nextseq 500. The sequencing reads were down-sampled to 2x1.2 million reads unless otherwise indicated, adapter trimmed and filtered with Prinseq. B. Shown are total down-sampled reads (Total reads), reads after adaptor trimming (Trimmed reads) and reads passing Prinseq filter (Filtered reads). Insets shown are example Bioanalyzer results for cDNA libraries made by each method. Non-specific products are highlighted in the dotted frame. C. Filtered reads were aligned to GENCODE 28 and ERCC transcripts using Salmon. Dots indicate the number of transcripts with TPM (Transcripts per million) ≥1 detected from each library as a function of sequencing depth.  D. Filtered reads were aligned to hg19 Human Reference Genome using Hisat 2.0.7 and RNA-seq metrics were calculated using Picard SAM/BAM RNA Seq Metrics tools. Shown are percentage of mapped reads distributed to exons (red), rRNAs (orange), introns (green), intergenic region (blue) and reads that cannot be mapped to the reference (gray).
 


Figure 3. Template Switching RT Enzyme Mix offers a simple workflow and superior performance for 5´ RACE.



A. Overview of template switching-mediated 5′ RACE. After the template switching reverse transcription reaction, 5′ RACE PCR is performed with a reverse gene-specific primer and a forward TSO-specific primer.
 
B. Agarose gel analysis of 5′ RACE products for various RNA targets using the NEB Template Switching RT Enzyme Mix 5′ RACE protocol (left) or Clontech SMARTer 5′/3′ RACE Kit (right). Input included 1 µg of Jurkat total RNA, 10 pg of 8 kb synthetic RNA and 10 ng of ERCC RNA Mix 1 to evaluate the performance as a function of transcript length and copy number. For the NEB reaction, oligo (dT)40 VN was used as an RT primer and GCTAATCATTGCAAGCAGTGGTATCAACGCAGAGTACATrGrGrG as the TSO, with the TSO-specific PCR primer underlined. The same internal gene-specific PCR primer was used for both methods. Target names and expected sizes are as indicated.
 


Figure 4. Template Switching RT Enzyme Mix offers a simple workflow for 2nd strand cDNA synthesis which captures the full 5′ end of transcripts.



A. Overview of template switching mediated 2nd strand cDNA synthesis. After the RT reaction, the RNA template is hydrolyzed and the 2nd strand cDNA is synthesized by primer extension using the TSO as a primer.
 
B. 1 kb synthetic RNA was used as template and poly(dT)40 VN was used as RT primer for 1st strand cDNA synthesis. Three independent experiments were performed for 2nd strand cDNA synthesis using either the template switching-mediated method or the method as described by Gubler. R and Hoffman, BJ. (1983) Gene, 25, 263-269. The resulting ds cDNA products were subject to Sanger Sequencing using a reverse primer to sequence the cDNA 2nd strand. The template sequence with the transcription start site (TSS) and the alignment consensus sequence are indicated. The TSO sequence added to the cDNA end is highlighted in gray. Sequences not reliably detected from the Gubler and Hoffman method are highlighted in blue. Bases with low confidence calls are highlighted in yellow.

This product is related to the following categories:
cDNA Synthesis & Reverse Transcriptases Products,
PCR, qPCR & Amplification Technologies Products,
This product can be used in the following applications:
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis)

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0466S     -20    
      Template Switching RT Enzyme Mix  M0466SVIAL -20 1 x 0.02 ml 10 X
      Template Switching RT Buffer B0466SVIAL -20 1 x 0.5 ml 4 X
  • M0466L     -20    
      Template Switching RT Enzyme Mix  M0466LVIAL -20 1 x 0.1 ml 10 X
      Template Switching RT Buffer B0466SVIAL -20 1 x 0.5 ml 4 X

Properties & Usage

Heat Inactivation

80°C

Protocols, Manuals & Usage

Protocols

  1. cDNA Synthesis and Amplification Protocol using the Template Switching RT Enzyme Mix (NEB #M0466)
  2. 2nd Strand cDNA Synthesis Protocol using the Template Switching RT Enzyme Mix (NEB #M0466)
  3. 5′ RACE Protocol using the Template Switching RT Enzyme Mix (NEB #M0466)

FAQs & Troubleshooting

FAQs

  1. Do I need to add RNase Inhibitor to the RT reaction when using the Template Switching RT Enzyme Mix?
  2. Numerous publications describe the addition of various additives (e.g., betaine and PEG) to improve template switching. Can I add any of those to my reaction?
  3. Is the Template Switching RT Enzyme Mix compatible with RT primers with degenerate bases for cDNA synthesis and amplification, as described in the mcSCRB method [1]? 
  4. What type of template switching oligos (TSOs) are compatible with the Template Switching RT Enzyme Mix?
     
  5. Can I use the Template Switching RT Enzyme Mix for regular RT reactions?
  6. What is the efficiency of template switching when using the Template Switching RT Enzyme Mix?
  7. Does the Template Switching RT Enzyme Mix work for prokaryotic RNA?
  8. Does the Template Switching RT Enzyme Mix work for fragmented RNA?
  9. What type of RT primers can be used with the Template Switching RT Enzyme Mix?
  10. Can I reduce the RT reaction time?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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