NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina®
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.
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.
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.
Use this tool to find citations related to NEBNext products. Search by product, keyword or instrument name – use the filters to select specific product areas.
Real E, et al. (2021) A single-cell atlas of Plasmodium falciparum transmission through the mosquito. Nat Commun; PubMedID: 34045457, DOI: 10.1038/s41467-021-23434-z
Quality, Safety & Legal
Quality Control Assays
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]
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 busdev@neb.com.
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 busdev@neb.com.
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