When seeing the full picture of small RNA species in a sample matters, there is only one library prep kit that truly delivers on the promise of being low bias. The NEBNext Low-bias Small RNA Library Prep Kit was designed to generate libraries that accurately reflect the relative proportions of RNA species present in a sample. The straightforward gel-free protocol with a wide input range makes this kit the fastest high-sensitivity kit for small RNA library prep on the market.
Lowest bias of all commercially available kits
Biologically relevant input range (1,000 ng – 0.5 ng Total RNA; 5 ng – 0.05 ng Enriched small RNA)
Faster, single-day workflow
Multiplexable, up to 480 LV Unique Dual Index primer pairs (available separately)
18-month shelf life
Note: The original NEBNext Small RNA Kits (NEB #E7300, E7580, E7560, E7330) are still available for purchase, although NEB recommends the NEBNext Low-bias Small RNA Library Prep Kit for enhanced accuracy, library quality, and improved workflow.
The NEBNext Low-bias Small RNA Library Prep Kit minimizes biased representation of small RNA species, more accurately reflecting the number and proportion of unique small RNAs present. NEB's next-generation kit does this via a randomized splint ligation-based approach (see below), which captures small RNAs (<120 nt) with a 5' phosphate and 3' hydroxyl group. Species identified include microRNAs (miRNAs) from a variety of samples (e.g., human, plant), transfer RNAs (tRNAs) and tRNA-derived fragments, small nucleolar RNAs (snoRNAs), and piwi-interacting RNAs (piRNAs). This kit is designed for the preparation of Illumina®-compatible small RNA sequencing libraries.
Benefits:
Analyze all RNA species present, without library prep-generated bias
Simplify and streamline your library prep workflow (~3.5 hours), saving time
Derive insights from biologically relevant input amounts
Plan ahead with a 18-month shelf life
Learn a single protocol for standard and 2'-O-methylated samples
Expand your insights with up to 480 compatible UDI primer pairs, available separately
The NEBNext Low-bias Small RNA Library Prep Kit was developed following the 2020 publication of original research work by NEB scientists in Nucleic Acids Research. The article, "A low-bias and sensitive small RNA library preparation method using randomized splint ligation" was authored by Sean Maguire, Greg Lohman, and Shengzi Guan. Several protocol enhancements have improved the kit to outperform the originally published methods.
This kit is compatible with NEBNext LV Unique Dual Index Primer Pairs Sets 1-5; see neb.com/oligos for more details.
Each kit component must pass rigorous quality control standards and, for each new lot, the entire set of reagents is functionally validated together by construction of an indexed library and sequencing on an Illumina sequencer.
Figure 1: NEBNext Low-bias Small RNA Library Prep workflow
Library construction with the NEBNext Low-bias Small RNA Library Prep Kit is robust and streamlined and can be completed in less than 4 hours. This enhanced workflow features protocol optimizations that enable shorter reaction times with two bead-purification steps that enable increased throughput and ease of use.
Figure 2: NEBNext Low-bias Small RNA Library Prep method
The NEBNext Low-bias Small RNA Library Prep Kit requires 1,000–0.5 ng of total RNA, or 5–0.05 ng of enriched small RNA. For small RNAs to be compatible with the kit, they must have 5´-monophosphate and 3´-hydroxyl ends to enable ligation to Illumina®-compatible adaptors. The workflow features a reagent-additive approach that requires only two bead purification steps. Initially, a 3´ adaptor is ligated to the RNA, and then excess adaptor molecules are removed. Next, two reactions happen simultaneously: the 5´ adaptor is ligated, and the previously ligated 3´ adaptor is enzymatically modified to create the reverse transcription primer. cDNA is then generated, and a bead-based size selection ensures removal of non-ligated adaptor molecules and larger cDNA molecules. PCR amplification makes full-length Illumina-sequenceable molecules and introduces i5 and i7 indices. There are two bead purification strategies that can be used after library amplification. One approach uses a bead size selection that results in overall enrichment of miRNAs. The other method uses bead purification, which results in a generalized small RNA library prep. The NEBNext Low-bias Small RNA Libraries are compatible with NEBNext LV Unique Dual Index Primer Pairs (Sets 1-5)..
Figure 3: NEBNext Low-bias Small RNA Library Prep Kit produces libraries with the lowest bias
NEBNext Low-bias Small RNA libraries were made using a mix of 100 synthetic control miRNAs, including five that had 3´ 2´-O-methyl ends. Libraries were prepared from 0.3 ng of the synthetic miRNA mix to compare library-generated bias between the NEBNext Low-bias Small RNA Library Prep Kit and small RNA kits from Revvity® (NEXTFLEX® Small RNA Sequencing Kit V4), Qiagen® (QIAseq® miRNA Library Kit) and Illumina (TruSeq® Small RNA Library Prep). Libraries were sequenced on an Illumina NextSeq® 500 (1 x 56 bases), and 5 million reads from each library were adaptor trimmed (Flexbar) and aligned to the synthetic sequences using STAR (v2.7.8a). Expected reads were calculated from total reads mapped to the synthetic controls, divided by the total number of control sequences, represented here by the black line at 100%. Percent of observed reads to expected reads was calculated for each control sequence and plotted across replicates. For the NEBNext libraries, 90% of the control mix miRNAs were located within 2-fold of expected, whereas the competitor kits had between 19–30% fall within that range. Competitors' kits had more underrepresented sequences (50–65%) than overrepresented (15-24%). Libraries produced by the NEBNext Low-bias Small RNA Kit reliably generated libraries with the lowest bias.
Figure 4: NEBNext Low-bias Small RNA Library Prep consistently identifies more miRNAs across a broad input range
NEBNext Low-bias Small RNA libraries consistently detect a higher number of miRNAs across the RNA input range when compared to libraries generated using competitors' kits, e.g., Revvity (NEXTFLEX Small RNA Sequencing Kit V4), Qiagen (QIAseq miRNA Library Kit) and Illumina (TruSeq Small RNA Library Prep). 1,000–0.5 ng of human brain total RNA (Takara® #636530) was used as input into each library prep protocol, according to each kit's input guidelines and protocol recommendations. NEBNext Low-bias Small RNA libraries were prepared (following the size selection protocol) after library amplification. Libraries were sequenced on Illumina NovaSeq® 6000 (1 x 56 bases; or 1 x 72 bases for Qiagen UMI) and 15 million reads were adaptor trimmed (Flexbar) and aligned to the hg38 genome with STAR (v2.7.8a). The STAR reference was built using gencode v35 main annotations, supplemented with gencode tRNA annotations, rRNA annotations for subunits not included in gencode, and piRNA annotations from piRNAdb v1.7.6 that did not overlap with other annotations. A. A cumulative-detection histogram for mapped miRNAs at different supporting read thresholds across all inputs and library prep kits indicates the number of different miRNAs detected by a range of kits. A dashed line marks the threshold of 10 supporting reads. The NEBNext Low-bias Small RNA libraries reliably detect the most miRNAs at all thresholds and inputs. B. A histogram of miRNAs detected at a threshold of 10 supporting reads for all kits and across all inputs is shown. Values represent three technical replicates, and error bars represent standard deviation. The NEBNext Low-bias Small RNA libraries identify the most miRNAs.
Figure 5: NEBNext Low-bias Small RNA Library Prep Kit identifies more unique 2'-O-methylated piRNAs from human testis
NEBNext Low-bias Small RNA libraries identifies 2'-O-methylated piRNAs in human testis. The graph indicates the number of mapped 2'-O-methylated piRNAs detected in human testis. The NEB libraries consistently identified more 2'-O-methylated piRNAs than competitors' kits. Small RNA libraries were made using 100 ng of human testis total RNA (BioChain® #R1234260-50) with the NEBNext Low-bias Small RNA Library Prep Kit (following the size selection protocol) or competitors' small RNA kits from Revvity (NEXTFLEX Small RNA Sequencing Kit V4) and Qiagen (QIAseq miRNA Library Kit). Libraries were sequenced on Illumina NovaSeq 6000 (1 x 56 bases; or 1 x 72 bases for Qiagen UMI), and 15 million reads were adaptor trimmed (Flexbar) and aligned to the hg38 genome with STAR (v2.7.8a). The STAR reference was built using gencode v35 main annotations, supplemented with gencode tRNA annotations, rRNA annotations for subunits not included in gencode, and piRNA annotations from piRNAdb v1.7.6 that did not overlap with other annotations.
Figure 6: NEBNext Low-bias Small RNA Library Prep Kit identifies more unique 2'O methylated miRNAs in plants
NEBNext Low-bias Small RNA libraries identify 2'-O-methylated miRNAs in plants. The graphs indicate the number of mapped 2'-O-methylated miRNAs found in samples of Arabidopsis, rice and soy. The NEB libraries consistently detected more 2'-O-methylated miRNAs than competitors' kits. Small RNA libraries were made using 100 ng of total RNA (Arabidopsis: BioChain #R1634310; rice: BioChain #R1634360; soy: BioChain #R1634370) with the NEBNext Low-bias Small RNA Library Prep Kit (following the size selection protocol) or competitors' small RNA kits from Revvity (NEXTFLEX Small RNA Sequencing Kit V4) and Qiagen (QIAseq miRNA Library Kit). Libraries were sequenced on Illumina NovaSeq 6000 (1 x 56 bases; or 1 x 72 bases for Qiagen UMI), and 15 million reads were adaptor trimmed (Flexbar) and aligned to their respective genomes (Arabidopsis: Arabidopsis thaliana TAIR 10; rice: IRGSP-1.0; soy: Glycine max v2.1). The STAR references for Arabidopsis, rice and soy were built with annotations from EnsemblPlants release 52, supplemented with miRNA annotations from miRbase for Arabidopsis and rice.
Figure 7: NEBNext Low-bias Small RNA Library Prep Kit prepares robust libraries from FFPE RNA, even at low inputs
NEBNext Low-bias Small RNA libraries were successfully produced using total RNA isolated from formaldehyde-fixed, paraffin-embedded (FFPE) samples of (A) human breast (BioChain Cat # R2235086; RIN 2.4 DV200 30-50%) and (B) human lung (BioChain #R2234152, RIN 2.6, DV200 <30%). RNA integrity of FFPE RNA was determined using the Agilent® Bioanalyzer® RNA 6000 Pico Kit. Robust libraries were generated using 100–1 ng of the input RNA following the bead size-selection protocol after library amplification. The data presented are the average of three technical replicates, and error bars indicate the standard deviation. Library profiles remain consistent between inputs run on an Agilent TapeStation® High Sensitivity D1000 tape.miRNAs detected from NEBNext Low-bias Small RNA libraries show high levels of reproducibility between replicates and input amounts. Small RNA libraries were made using 100–1 ng of total RNA from formaldehyde-fixed, paraffin-embedded (FFPE) samples of human breast and human lung, following the bead size selection protocol after library amplification. Libraries were sequenced on Illumina NovaSeq 6000 (1 x 56 bases), and 15 million reads were adaptor trimmed (Flexbar) and aligned to the hg38 genome with STAR (v2.7.8a). The STAR reference was built using gencode v35 main annotations supplemented with gencode tRNA annotations, rRNA annotations for subunits not included in gencode, and piRNA annotations from piRNAdb v1.7.6 that did not overlap with other annotations. Correlations for two replicates of each input for lung and breast FFPE libraries were generated. Each point represents an individual miRNA, where axes are on a log scale, and R2 values indicate linear fit.
More data supporting the claims made about the NEBNext Low-bias Small RNA Library Prep Kit can be found on the E3420 Supplemental Data Page.
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