Product Class:

NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina®
Item# Description List Price Web Price Qty
E6420L NEBNext Single Cell Low Input RNA Library Prep Kit for Illumina - 96 rxns $5,630.00
$5,067.00
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E6420S NEBNext Single Cell Low Input RNA Library Prep Kit for Illumina - 24 rxns $1,697.00
$1,527.30
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

    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

    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 adaptors and 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.

    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
    NEBNext® Adaptor Dilution Buffer -20
    TE Buffer -20
    Nuclease-free Water -20
    NEBNext® Ligation Enhancer -20
    NEBNext® Ultra II Ligation Master Mix -20
    NEBNext® Ultra II Q5® Master Mix -20 2X
    NEBNext® Ultra II FS Enzyme Mix -20
    NEBNext® Ultra II FS Reaction Buffer -20
    Product Categories:
    Sample Prep for NGS & Target Enrichment Products,
    RNA Library Prep Products
    Applications:
    RNA Library Preparation,
    RNA For Illumina,
    Sample Prep for NGS & Target Enrichment
    ,
    Illumina Library Preparation

    Properties & Usage

    Materials Required but not Supplied

    80% Ethanol (freshly prepared)
    Nuclease-free Water
    DNA LoBind Tubes (Eppendorf® #022431021)
    Magnetic rack/stand (Alpaqua®, cat. #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

    Protocols

    1. Protocol for Cells: cDNA Synthesis, Amplification and Library Generation (E6420)
    2. Protocol for Low Input RNA: cDNA Synthesis, Amplification and Library Generation (E6420)

    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 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?

    Troubleshooting

    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

    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.

    Licenses


    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.