Next® Single Cell/Low Input RNA Library Prep Kit for Illumina® |NEB酶试剂 New England Biolabs

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产品信息

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


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

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


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

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


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

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 (2×76 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


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

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 (2×76 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


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

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 (2×76 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.

产品类别:
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • 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

  • 特性和用法

    需要但不提供的材料

    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)

操作说明、说明书 & 用法

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE6420

FAQs & 问题解决指南

  • 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)?