Product Class: Kit

NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina®

Product Introduction

How low can you go?

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

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

  • Generate the highest yields of high-quality sequencing libraries 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
Catalog # Size Concentration
E6420S 24.0 reactions
E6420L 96.0 reactions

Product Information

Description

The NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® uses a template switching method to generate full length cDNAs directly from single cells or 2 pg – 200 ng RNA, followed by conversion to sequence-ready libraries using the Ultra™ II FS workflow. This unique workflow enables generation of the highest yields from a broad range of inputs, and superior transcript detection, while providing reliably consistent performance.

Please note that NEBNext Multiplex Oligos (Adaptors & Primers) are not included in the kit and are available separately. 

Features:

  • The highest yields of high-quality sequencing libraries from single cells, or 2 pg - 200 ng RNA
  • Input can be cultured or primary cells, or total RNA
  • Low abundance transcripts are easily detected
  • High quality libraries and sequence data are generated for a wide range input amounts and sample types
    • Full length transcript coverage
    • Consistent transcript detection
    • Superior transcript correlation
  • Fast, streamlined, automation-friendly workflow, with minimal handling steps and hands-on time
    • Single-tube protocol from cell lysis to cDNA
    • DNA fragmentation, end repair and dA-tailing reagents in a single enzyme mix, with a single protocol, regardless of GC content

 

Download extensive performance data in our technical note.

 

Figure 1: NEBNext Single Cell/Low Input RNA Library Prep workflow


Figure 2: Higher library yields with the NEBNext Single Cell/Low Input RNA Library Prep Kit




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 RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® #634891) plus the Nextera® XT DNA Library Prep Kit (Illumina® #FC-131-1096) were used. Error bars indicate standard deviation for 6-11 replicates. For the NEBNext workflow ~80% of the cDNA was used as input into sequencing library preparation, and libraries were amplified with 8 PCR cycles. For the SMART-Seq®v4/Nextera XT workflow, as recommended, 125 pg of cDNA was used as input in sequencing library preparation and 12 PCR cycles were used for amplification. Error bars indicate standard deviation for 6-11 replicates. 
Figure 3: Increased transcript detection with the NEBNext Single Cell/Low Input RNA Library Prep Kit




Sequencing libraries were generated from Jurkat single cells (6 replicates) using the NEBNext Single Cell/Low Input RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® # 634891) plus the Nextera XT DNA Library Prep Kit (Illumina® #FC-131-1096). Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). TPM = Transcripts per Kilobase Million. Each dot represents the number of transcripts identified at the given TPM range, and each box represents the median, first and third quartiles per replicate and method. Salmon 0.6 was used for read mapping and quantification of all GENCODE v25 transcripts. Panels show the number of transcripts detected within the following TPM ranges: 1-5, 5-10, 10-50 and >50 TPM. Increased identification of low abundance transcripts is observed with the NEBNext libraries.
Figure 4: The NEBNext Single Cell/Low Input RNA Library Prep Kit provides uniform coverage across the length of transcripts




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 RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® # 634891) plus the Nextera XT DNA Library Prep Kit (Illumina® #FC-131-1096) were used. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Gene body coverage shown is an average of four replicates and was calculated using Picard tools. The global view of the 5´ to 3´ coverage of the RefSeq transcripts reveals both consistency across different sample types and uniformity across the transcript length in the NEBNext libraries.
Figure 5: Low input UHR RNA libraries retain complexity with the NEBNext Single Cell/Low Input RNA Library Prep Kit




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 RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® # 634891) plus the Nextera XT DNA Library Prep Kit (Illumina #FC-131-1096) were used. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Error bars indicate standard deviation for 6-11 replicates. TPM = Transcripts per Kilobase Million. Salmon 0.6 was used for read mapping and quantification of all GENCODE v25 transcripts. A higher number of transcripts were detected in the NEBNext libraries for all sample types.

View additional performance data in our technical note.
This product is related to the following categories:
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E6420S     -20    
      NEBNext® Single Cell RT Primer Mix E6422AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Single Cell RT Buffer E6423AVIAL -20 1 x 0.12 ml Not Applicable
      NEBNext® Template Switching Oligo E6424AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext® Single Cell RT Enzyme Mix E6425AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Single Cell cDNA PCR Master Mix E6426AVIAL -20 1 x 1.2 ml Not Applicable
      NEBNext® Single Cell cDNA PCR Primer E6427AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Cell Lysis Buffer E6428AVIAL -20 1 x 0.048 ml Not Applicable
      Murine RNase Inhibitor E6429AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext® Bead Reconstitution Buffer E6430AVIAL -20 1 x 1.08 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E6431AVIAL -20 1 x 0.576 ml Not Applicable
      TE Buffer E6432AVIAL -20 1 x 1.2 ml Not Applicable
      Nuclease-free Water E6433AVIAL -20 1 x 1.44 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AVIAL -20 1 x 0.6 ml Not Applicable
      NEBNext® Ultra™ II FS Enzyme Mix E7806AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Ultra™ II FS Reaction Buffer E7807AVIAL -20 1 x 0.168 ml Not Applicable
  • E6420L     -20    
      NEBNext® Single Cell RT Primer Mix E6422AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Single Cell RT Buffer E6423AAVIAL -20 1 x 0.48 ml Not Applicable
      NEBNext® Template Switching Oligo E6424AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext® Single Cell RT Enzyme Mix E6425AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Single Cell cDNA PCR Master Mix E6426AAVIAL -20 1 x 4.8 ml Not Applicable
      NEBNext® Single Cell cDNA PCR Primer E6427AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Cell Lysis Buffer E6428AAVIAL -20 1 x 0.192 ml Not Applicable
      Murine RNase Inhibitor E6429AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext® Bead Reconstitution Buffer E6430AAVIAL -20 1 x 4.32 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E6431AAVIAL -20 1 x 2.31 ml Not Applicable
      TE Buffer E6432AAVIAL -20 1 x 4.8 ml Not Applicable
      Nuclease-free Water E6433AAVIAL -20 1 x 5.76 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AAVIAL -20 2 x 1.2 ml Not Applicable
      NEBNext® Ultra™ II FS Enzyme Mix E7806AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Ultra™ II FS Reaction Buffer E7807AAVIAL -20 1 x 0.672 ml Not Applicable

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)
Thermocycler
Vortex
Microcentrifuge
SPRIselect Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP
Beads (Beckman Coulter, Inc. #A63881)
Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables
NEBNext Oligos
DNase RNase free PCR strip tubes (USA Scientific 1402-1708)

Protocols, Manuals & Usage

Manuals

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

FAQs & Troubleshooting

FAQs

  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 RNA Library Prep Kit 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 (prior to cDNA amplification) 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 kit?
  11. Are the two PCR master mixes in the kit interchangeable?
  12. For low cDNA input do I have to do a double bead cleanup after the final PCR?
  13. How much cDNA input can I use for the NEBNext Ultra II FS DNA library preparation?
  14. What is the expected size range of libraries generated with the NEBNext Single Cell/Low Input RNA Library Prep Kit?
  15. Why are the adaptor dilution and library amplification PCR cycle number recommendations in this kit different from the NEBNext Ultra II FS DNA Library Prep Kit?
  16. Does the NEBNext Single Cell/Low Input RNA Library Prep Kit include adaptors and primers?
  17. How should the adaptor sequences be trimmed?
  18. Which NEBNext Oligos can be used with this library prep kit?
  19. Can I use this NEBNext kit with adaptors and primers from other vendors than NEB?
  20. Where can I find protocols for low-input RNA library prep for use with the following kits: NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB #E6420), NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB #E6421), and NEBNext® Single Cell Lysis Module (#E5530)?

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.

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 [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.

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.

Licenses

This product is licensed for research and commercial use 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. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

For additional information or to inquire about commercial use, please contact [email protected].

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