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

Catalog # Concentration Size List Price Quantity Your Price
E6420L 96 reactions $6,308.00
$5,677.20
E6420S 24 reactions $1,901.00
$1,710.90
Catalog # Size List Price Your Price
E6420L 96 reactions $6,308.00
$5,677.20
E6420S 24 reactions $1,901.00
$1,710.90
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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

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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  NEBNext® Single Cell RT Primer Mix E6422AVIAL -20 1 x 0.048 ml
  NEBNext® Single Cell RT Buffer E6423AVIAL -20 1 x 0.12 ml
  NEBNext® Template Switching Oligo E6424AVIAL -20 1 x 0.024 ml
  NEBNext® Single Cell RT Enzyme Mix E6425AVIAL -20 1 x 0.048 ml
  NEBNext® Single Cell cDNA PCR Master Mix E6426AVIAL -20 1 x 1.2 ml
  NEBNext® Single Cell cDNA PCR Primer E6427AVIAL -20 1 x 0.048 ml
  NEBNext® Cell Lysis Buffer E6428AVIAL -20 1 x 0.048 ml
  Murine RNase Inhibitor E6429AVIAL -20 1 x 0.024 ml
  NEBNext® Bead Reconstitution Buffer E6430AVIAL -20 1 x 1.08 ml
  NEBNext® Adaptor Dilution Buffer E6431AVIAL -20 1 x 0.576 ml
  TE Buffer E6432AVIAL -20 1 x 1.2 ml
  Nuclease-free Water E6433AVIAL -20 1 x 1.44 ml
  NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml
  NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml
  NEBNext® Ultra II Q5® Master Mix E7649AVIAL -20 1 x 0.6 ml
  NEBNext® Ultra™ II FS Enzyme Mix E7806AVIAL -20 1 x 0.048 ml
  NEBNext® Ultra™ II FS Reaction Buffer E7807AVIAL -20 1 x 0.168 ml
  NEBNext® Single Cell RT Primer Mix E6422AAVIAL -20 1 x 0.192 ml
  NEBNext® Single Cell RT Buffer E6423AAVIAL -20 1 x 0.48 ml
  NEBNext® Template Switching Oligo E6424AAVIAL -20 1 x 0.096 ml
  NEBNext® Single Cell RT Enzyme Mix E6425AAVIAL -20 1 x 0.192 ml
  NEBNext® Single Cell cDNA PCR Master Mix E6426AAVIAL -20 1 x 4.8 ml
  NEBNext® Single Cell cDNA PCR Primer E6427AAVIAL -20 1 x 0.192 ml
  NEBNext® Cell Lysis Buffer E6428AAVIAL -20 1 x 0.192 ml
  Murine RNase Inhibitor E6429AAVIAL -20 1 x 0.096 ml
  NEBNext® Bead Reconstitution Buffer E6430AAVIAL -20 1 x 4.32 ml
  NEBNext® Adaptor Dilution Buffer E6431AAVIAL -20 1 x 2.31 ml
  TE Buffer E6432AAVIAL -20 1 x 4.8 ml
  Nuclease-free Water E6433AAVIAL -20 1 x 5.76 ml
  NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml
  NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml
  NEBNext® Ultra II Q5® Master Mix E7649AAVIAL -20 2 x 1.2 ml
  NEBNext® Ultra™ II FS Enzyme Mix E7806AAVIAL -20 1 x 0.192 ml
  NEBNext® Ultra™ II FS Reaction Buffer E7807AAVIAL -20 1 x 0.672 ml
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

DataCards
FAQs
Citations
Additional Citations
  • Mamanova L, et al. (2021) High-throughput full-length single-cell RNA-seq automation. Nat Protoc DOI: 10.1038/s41596-021-00523-3
  • Real E, et al. (2021) A single-cell atlas of Plasmodium falciparum transmission through the mosquito. Nat CommunPubMedID: 34045457, DOI: 10.1038/s41467-021-23434-z
Quality Control Assay
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Certificate of Analysis
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Safety Data Sheets
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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.

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 busdev@neb.com.

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