Product Class:

NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module
Item# Description List Price Web Price Qty
E6421L NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module - 96 rxns $3,201.00
E6421S NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module - 24 rxns $941.00
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at

Product Introduction

How low can you go?

NEBNext® is now available for single cell and ultra-low RNA inputs!

This unique cDNA synthesis and amplification workflow meets the demand for a highly sensitive, yet robust method that consistently ultimately enables generation of high quality sequencing data from single cell or ultra-low input RNA.

  • Generate the highest yields from single cells, or as little as 2 pg - 200 ng RNA
  • Experience unmatched detection of low abundance transcripts
  • Rely on consistent transcript detection for a wide range of input amounts and sample types
  • Observe uniform transcript coverage, regardless of input amount or sample type
  • Use with a variety of RNA inputs, including cultured or primary cells, or total RNA
  • Save time with a fast, streamlined workflow, minimal handling steps and hands-on time

Please note that this module contains only reagents for cDNA synthesis and amplification. The NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB #E6420) includes reagents for DNA library construction.

Product Information


The NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module uses a template switching method to generate full length cDNAs directly from single cells or 2 pg – 200 ng RNA. This unique workflow enables generation of high quality sequencing data from a broad range of inputs, and superior transcript detection, while providing reliably consistent performance.


  • The highest yields from single cells, or 2 pg - 200 ng RNA
  • Input can be cultured or primary cells, or total RNA
  • Single-tube protocol from cell lysis to cDNA
  • Fast, streamlined, automation-friendly workflow, with minimal handling steps and hands-on time

Download extensive performance data in our technical note.

Figure 1: NEBNext Single Cell/Low Input workflow

Figure 2: Higher cDNA yields with the NEBNext Single Cell/ Low Input cDNA Synthesis & Amplification Module

Sequencing libraries were generated from HeLa, Jurkat and M1 single cells, or 10 pg of Universal Human Reference (UHR) RNA (Agilent® #740000) with recommended amounts of ERCC RNA Spike-In Mix I (Thermo Fisher Scientific® #4456740). The NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module or the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® # 634891) were used. cDNA libraries were amplified using the number of PCR cycles shown. Error bars indicate standard deviation for 8-21 replicates.

View extensive performance data i in our technical note.

For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact [email protected] for further information.

Each set of reagents is functionally validated together through construction and sequencing of a transcriptome library and sequenced on a

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C) Concentration
NEBNext® Single Cell RT Primer Mix -20
NEBNext® Single Cell RT Buffer -20
NEBNext® Template Switching Oligo -20
NEBNext® Single Cell RT Enzyme Mix -20
NEBNext® Single Cell cDNA PCR Master Mix -20
NEBNext® Single Cell cDNA PCR Primer -20
NEBNext® Cell Lysis Buffer -20
Murine RNase Inhibitor -20
NEBNext® Bead Reconstitution Buffer -20
TE Buffer -20
Nuclease-free Water -20
Product Categories:
Sample Prep for NGS & Target Enrichment Products,
RNA Library Prep Products
RNA For Illumina,
RNA Library Preparation,
Sample Prep & Target Enrichment for NGS
Illumina Library Preparation

Properties & Usage

Materials Required but not Supplied

80% Ethanol (freshly prepared)
Nuclease-free Water
DNA LoBind Tubes (Eppendorf® #022431021)
Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
SPRIselect Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP
Beads (Beckman Coulter, Inc. #A63881)
Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables
DNase RNase free PCR strip tubes (USA®  Scientific 1402-1708)

Protocols, Manuals & Usage


  1. Protocol for Cells: cDNA Synthesis and Amplification (NEB# E6421)
  2. Protocol for Low Input RNA: cDNA Synthesis and Amplification (NEB #E6421)


The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Faqs & Troubleshooting


  1. How much PBS can be carried over from cell collection into the cDNA Synthesis and Amplification protocol?
  2. What cell types does the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module support?
  3. Can I resuspend the cells in buffers/medium other than PBS before cell collection?
  4. Do the cells have to be sorted in to a specific reagent?
  5. Can frozen cells be used?
  6. Can cells be lysed using other cell lysis buffers?
  7. How long can the synthesized cDNA be stored for prior to amplification?
  8. What types of RNA are compatible?
  9. Does the input total RNA have to be purified?
  10. My total RNA sample is in a volume > 8 µl. Can I still use the module?
  11. Is the cDNA generated by the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification protocol compatible with Nextera® XT from Illumina?
  12. How should the adaptor sequences be trimmed?


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.


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

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

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.


This product is licensed from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645 and corresponding patents in other countries for use only in: (a) standard (not real-time) PCR in the research field only, but not real time PCR or digital PCR; (b) real-time PCR for use as a library preparation quantitation tool in Next Generation Sequencing workflows; (c) any in-vitro diagnostics applications, except for applications using real-time PCR or digital PCR; and (d) any non-PCR applications in DNA sequencing, isothermal amplification, and the production of synthetic DNA.