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.
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
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.
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)
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
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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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