AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq)

Template-Switching Enabled Full-Length cDNA Synthesis for High-Fidelity Single-Cell Transcriptomics

Full-length cDNA

Template-switching reverse transcription

88% GC stem-loop

Validated on high-GC RNA

Engineered RT

Higher template switching than competitors

Product overview

The AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq) is optimized for high-throughput, full-length first-strand cDNA synthesis from mammalian or cell-wall-free eukaryotic cells. It leverages a genetically engineered reverse transcriptase and a streamlined template-switching workflow to preserve 5′ end sequence information of mRNA, delivering cDNA products with defined known sequences at both termini for downstream library construction.

Features and applications

Features

Full-Length cDNA Synthesis via Template Switching: The engineered reverse transcriptase initiates reverse transcription by binding an oligo(dT) primer to the poly(A) tail of mRNA, then adds a 3-cytosine (CCC) tail to the 3′ end of the nascent cDNA upon reaching the mRNA 5′ end. This poly(C) tail serves as an anchor for the pre-added Template-Switching Oligo (TSO), whose 3′ end contains three guanines (GGG) complementary to the CCC tail. The reverse transcriptase then performs template switching and extends to the end of the TSO, generating full-length cDNA with standardized termini.
Engineered Reverse Transcriptase Superiority: Compared to wild-type M-MLV reverse transcriptase, the engineered enzyme delivers markedly improved thermal stability, superior reverse-transcription efficiency, and enhanced tolerance to high DTT concentrations, ensuring robust performance across diverse reaction conditions.
Broad Input Compatibility: Compatible with 10 pg to 1 μg of poly(A)-containing total RNA, supporting both single-cell and low-input bulk RNA workflows.
Seamless Downstream Integration: The resulting full-length cDNA with defined terminal sequences is directly compatible with standard downstream workflows including cDNA amplification, enzymatic fragmentation, and sequencing library preparation.

Applications

High-throughput single-cell 3′ RNA sequencing (scRNA-seq)
Transcriptomic profiling of rare cell populations (e.g., circulating tumor cells, antigen-specific immune cells)
Low-input transcriptome analysis from limited eukaryotic cell samples
Full-length cDNA library construction for transcript isoform discovery

Components

Component	Specification/Notes
4×RT Buffer	4×
Reverse Transcriptase	/

Performance data

Temperature-stable Reverse Transcriptase Performance

BR3N801 AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq): Figure 1. Reverse transcriptase activity comparison across temperatures — **Figure 1. Reverse transcriptase activity comparison across temperatures.** BR3N801 (the core component of the AcuGenix™ First-Strand cDNA Synthesis Kit) and Supplier T were evaluated in parallel at 40°C, 50°C, and 55°C. Specific activity (U/μL) was measured for each condition. BR3N801 demonstrated activity parity with Supplier T, with slight gains at 40°C and 55°C, and equivalent performance at 50°C. Thermal stability is a critical determinant of full-length cDNA synthesis efficiency in single-cell workflows, as elevated reaction temperatures minimize secondary structure interference.

Group	Reverse Transcriptase (RT)	Enzyme Activity (U/μL)
1	Supplier T	895	1472	1972
3	BR3N801	957.25	1399.94	2077.67

Note: BR3N801 is the catalog number for this kit. Higher thermal tolerance reduces amplification bias for GC-rich or structured RNA templates.

High-efficiency Template Switching

BR3N801 AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq): Figure 2. Improved template switching efficiency of BR3N801 — **Figure 2. Improved template switching efficiency of BR3N801.** Single RNA templates were reverse-transcribed using BR3N801 or Supplier T RTase via the C-tailing and Template-Switching Oligo (TSO) capture workflow. qPCR yielded a Ct of 4.99 for BR3N801 versus 5.26 for Supplier T. Lower Ct values correspond to higher cDNA yield and more complete template switching, confirming superior performance for low-input and single-cell applications.

Note: TSO (Template-Switching Oligo) is the core reagent enabling full-length cDNA synthesis in single-cell 3′ RNA-seq. This assay mimics real-world library preparation conditions.

High GC and Stem-loop Compatibility

BR3N801 AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq): Figure 3. Enhanced compatibility with high-GC stem-loop RNA — **Figure 3. Enhanced compatibility with high-GC stem-loop RNA.** BR3N801 and Supplier T were evaluated using an 88% GC stem-loop RNA template via the C-tailing/TSO capture workflow. ΔCt (calculated as Ct_outer primer – Ct_inner primer) was 1.67 for Supplier T and 0.42 for BR3N801. Lower ΔCt values indicate reduced amplification bias against folded RNA structures, confirming BR3N801’s superior tolerance for challenging templates.

Note: Stem-loop structures are a common cause of RT arrest; ΔCt is the standard metric for evaluating secondary structure tolerance.

Higher Switching Efficiency

BR3N801 AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq): Figure 4. Higher template switching efficiency than competitors — **Figure 4. Higher template switching efficiency than competitors.** BR3N801, Supplier Y, and Supplier T were tested on human total RNA using a standard C-tailing/TSO workflow. All three enzymes produced identical fragment size distributions with no length bias. BR3N801 generated a stronger full-length cDNA signal, reflecting higher template switching conversion efficiency and more complete preservation of 5′ mRNA sequence information.

Drop-in Equivalence to Supplier G Full Workflow

BR3N801 AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq): Figure 5. Drop-in equivalence to the Supplier G full system — **Figure 5. Drop-in equivalence to the Supplier G full system.** BR3N801 was substituted for the reverse transcriptase component in the Supplier G kit without protocol modification. Top: cDNA yield and fragment distribution were concordant with the native Supplier G system. Bottom: Post-sequencing cell cluster profiles and gene counts showed high concordance with Supplier G results. BR3N801 maintains end-to-end workflow integrity as a seamless alternative requiring no protocol re-optimization.

BR3N801 AcuGenix™ First-Strand cDNA Synthesis Kit (for Single-Cell 3′ RNA-Seq): Figure 5 (panel 2) drop-in equivalence to the Supplier G full workflow — **Figure 5. Drop-in equivalence to the Supplier G full system.** BR3N801 was substituted for the reverse transcriptase component in the Supplier G kit without protocol modification. Top: cDNA yield and fragment distribution were concordant with the native Supplier G system. Bottom: Post-sequencing cell cluster profiles and gene counts showed high concordance with Supplier G results. BR3N801 maintains end-to-end workflow integrity as a seamless alternative requiring no protocol re-optimization.

Note: Drop-in equivalence is an industry-standard metric confirming that a component can replace an existing reagent without altering workflow performance.

Enhanced scRNA-seq Recovery

Note: Genes per cell and UMI yield are core metrics for scRNA-seq sensitivity; higher values indicate more complete transcriptome capture at the single-cell level.