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 OverviewUpon 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-seqA. 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 GCTAATCATTGCAAGCAGTGGTATCAACGCAGAGTACATrGrGrGas 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:
购物车为空，快去购物吧！
4000-520-616
咨询顾问
特别 提示
