NEBNext UltraExpress™ RNA: A fast and flexible workflow
for stranded RNA-seq library preparation
Chelsea Pinegar, Anetta Nowosielska-Vecchietti, Gautam Naishadham, Chen Song, Bradley
W. Langhorst, and Keerthana Krishnan | New England Biolabs, Inc.

Introduction
RNA sequencing (RNA-seq) has become an invaluable tool in the study of biology, but often
requires multiple days to process samples from total RNA to final libraries. When working with samples
in a high-throughput capacity in particular, protocol transfer to automation and time to prepare librariescan be a great hindrance for processing samples efficiently.
The NEBNext UltraExpress™ RNA Library Prep Kit provides a new protocol that allows users toeasily generate robust RNA-seq libraries from a variety of sample types, including fragmented RNA, ina single day from total RNA. Compatible with poly(A) enrichment or ribosomal RNA depletion, theworkflow has reduced preparation time by ~40% compared to leading library preparation kits, without
sacrificing library quality or sequencing metrics. In addition to faster preparation times, this kit provides
flexibility to users, with a protocol that is automation friendly and allows implementation of a single
protocol for all RNA inputs within the recommended range. In addition to fewer components and shorter
incubation times, the reduction of bead cleanup steps significantly reduces hands-on time, simplifying
and streamlining the library preparation process.
Libraries for RNA-seq were prepared for Illumina® sequencing using several commercially-availablereferences, including Universal Human Reference RNA, a bacterial RNA mix, total RNA extracted fromhuman whole blood (HWB) and several plant RNA samples. Here, we demonstrate how the NEBNext
UltraExpress RNA library preparation workflow provides a great advantage for efficiently generating
strand-specific RNA-seq libraries while simultaneously maintaining high-quality sequencing results,
including 5’-3’ transcript coverage, excellent transcript detection, and high correlation of transcript
expression across replicates and inputs.

Methods

Results (continued)
Consistent 5’-3’ transcript coverage

5´ to 3´ transcript coverage was calculated from the top 1,000 transcripts using the CollectRnaSeqMetrics (Picard) tool v2.18.2.2 usingreads mapped to the hg38 reference genome using RNA STAR v2.7.8a. (A) Libraries were prepared from poly(A)-enriched UHRR using
the NEBNext UltraExpress RNA Library Prep Kit, Kapa mRNA HyperPrep Kit, or Illumina Stranded mRNA Kit. (B) Libraries were preparedfrom UHRR using the UltraExpress RNA Library Prep Kit preceded by the NEBNext rRNA Depletion Kit, KAPA HyperPrep Kit with
RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero Plus.

NEBNext UltraExpress RNA Library Prep

Excellent transcript correlation between inputs and replicates

The NEBNext UltraExpress RNA Library Prep Kit (NEB #E3330) features a fast, streamlined
workflow, with a 3-hour library prep protocol that can produce high-quality directional RNA libraries inconjunction with mRNA enrichment or rRNA depletion in a single day.

The NEBNext UltraExpress RNA Library Prep Kit uses a single adaptor dilution (50X) and 12(poly(A)-enriched) or 11 (ribodepleted) PCR cycles for all inputs. Alternatively, users can use
customized adaptor dilutions (20X for 76–250ng, 100X for 25–75 ng) and PCR cycle numbers (14/13(poly(A)/ribodepleted) for 25 ng inputs, 12/11 for 75 ng inputs, or 10/9 for 250 ng inputs) for each input.
For these results, libraries were prepared from Universal Human Reference RNA (UHRR, Agilent®)
containing External RNA Control Consortium (ERCC) Spike-In Controls (Invitrogen™), unless otherwise
noted, and enriched using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490),
NEBNext rRNA Depletion Kit (Human/Mouse/Rat)(NEB #E7400), or other protocol as specified.
Libraries were sequenced on an Illumina NextSeq® 500 (2 x 75 bases) and 10 million reads were
sampled from each library for analysis.

Results
High library yields across a range of inputs

Libraries were prepared from UHRR using the NEBNext UltraExpress RNA Library Prep Kit with poly(A) enrichment (A) or ribosomal RNA(rRNA) depletion (B). Correlation of transcript abundance was calculated across inputs using Salmon v1.5.1 quantification of all gencodev38 transcripts and ERCCs. Each data point represents a transcript, with the log10 abundance of reads from 250 ng total RNA input
compared to a replicate of 250 ng as well as 75 and 25 ng inputs.

Excellent transcript correlation across sample types(A) RNA from human whole blood (HWB) was depleted of globin mRNA and rRNA using the NEBNext Globin & rRNA Depletion Kit(Human/Mouse/Rat)(NEB #E7750). (B) rRNA was depleted from a pool of 20 different bacterial organisms (ATCC® #MSA-2002) using theNEBNext rRNA Depletion Kit (Bacteria)(NEB #E7850). Libraries were prepared using the NEBNext UltraExpress RNA Library Prep Kit andtranscript expression levels were correlated across inputs using Salmon v1.5.1 quantification of all gencode v38 transcripts (HWB) and acomposite genome (Bacterial). Each data point represents a transcript, with the log10 abundance in number of reads with 250 ng total RNA
input on the y-axis compared to a replicate at 250 ng followed by 75 and 25 ng on the x-axis.

Robust ribodepletion and transcript detection from plants
Oryza sativa

94.5%
Rice_undepl_250ng_R2% rRNA

80.0%

High sensitivity of transcript detection

60.0%

Rice_250ng_R5 0.8%

25ng

Rice_250ng_R2

2.6%
Rice_250ng_R1

Rice_25ng_R3 1.5%

4.4%
Rice_25ng_R1

4.5%

2.6%

Corn_250ng_R1

Corn_250ng_R3 2.2%

3.9%
Corn_25ng_R4

250ng

25ng

Corn_250ng_R2

4.6%
Corn_25ng_R3

Corn_25ng_R2 1.8%250ng

Corn_25ng_R1

2.4%

5.3%

Ara_250ng_R2

A.thaliana

Ara_250ng_R3

3.7%

2.4%

Ara_250ng_R1

Ara_25ng_R3 1.9%

3.0%
Ara_25ng_R225ng

Corn_undepl_250ng_R2

25ng depleted (Read counts log10)

B.

4.7%

0.0%

Ara_25ng_R1

20.0%

6.6%

40.0%

250ng

Z. maysAra_25ng_R2
Ara_25ng_R3
Ara_250ng_R1
Ara_250ng_R2Ara_250ng_R3
Ara_undepl_25ng_R1
Ara_undepl_25ng_R2
Ara_undepl_250ng_R1
Ara_undepl_250ng_R2
Corn_25ng_R1
Corn_25ng_R2
Corn_25ng_R3
Corn_25ng_R4
Corn_250ng_R1
Corn_250ng_R2
Corn_250ng_R3
Corn_undepl_25ng_R1
Corn_undepl_25ng_R2
Corn_undepl_250ng_R1
Corn_undepl_250ng_R2
Rice_25ng_R1
Rice_25ng_R3
Rice_250ng_R1
Rice_250ng_R2
Rice_250ng_R5
Rice_undepl_25ng_R1
Rice_undepl_25ng_R2
Rice_undepl_250ng_R1
Rice_undepl_250ng_R2

Total RNA from A. thaliana, Z. mays, and O. sativa (Biochain) was
depleted of ribosomal RNA (rRNA) using a custom plant probe poolwith the NEBNext RNA Depletion Core Reagent Set (NEB #E7865).
Read pairs were identified as ribosomal using mirabait (six or more25-mers). Transcript abundances were estimated using Salmon(v1.3.0) to correlate results from low and high input libraries.
Transcripts were obtained from annotations of transcribed featuresin Ensembl release 45 (TAIR10 for A. thaliana, Zm-B73 Refgen V2for Z. mays and IRGSP-1.0 for O. sativa).

Sum of % rRNA for each Library. Color shows details about Library. The marks are labeled by % rRNA as an attribute. The context isfiltered on Id (Workflow Invocations) Set, which keeps 78 members. The data is filtered on Category, which keeps PAIR andUNPAIRED. The view is filtered on Library, which excludes 7 members.

R2=0.996
R2=0.999

250ng depleted (Read counts log10)

Z. maysR2=0.981
R2=0.986
250ng depleted (Read counts log10)

25ng depleted (Read counts log10)

94.3%
Rice_undepl_250ng_R1

95.4%

25ng depleted (Read counts log10)Corn_undepl_250ng_R1

95.7%

95.6%
Corn_undepl_25ng_R2

94.2%
Ara_undepl_250ng_R2

undepleted
95.6%

94.0%
Ara_undepl_250ng_R1

Library
depleted

Corn_undepl_25ng_R1

94.3%
Ara_undepl_25ng_R2

100.0%

94.1%

undepleted

Ara_undepl_25ng_R1

depleted

94.5%

rRNA (2)(A) rRNA sequencing reads percentage in libraries with and withoutrRNA depletion. (B) Transcript detection correlation between 25ng
depleted undepleted Libraryand 250ng input.
Ara_25ng_R1

Rice_undepl_25ng_R2

Zea Mays

94.4%Library yields were measured using an Agilent 4200 TapeStation. Shown are averages of triplicates with error bars indicating the standard
deviation. Total RNA input mass and number of PCR cycles are indicated. (A) Libraries were prepared from poly(A)-enriched UHRR using
the NEBNext UltraExpress RNA Library Prep Kit, Kapa mRNA HyperPrep® Kit, or Illumina Stranded mRNA Kit. (B) Libraries were preparedfrom UHRR using the UltraExpress RNA Library Prep Kit preceded by the NEBNext rRNA Depletion Kit, Kapa HyperPrep Kit withRiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero® Plus.

Arabidopsis thaliana

Rice_undepl_25ng_R1A.

O.sativa

R2=0.980
R2=0.997

250ng depleted (Read counts log10)

Conclusions

The average number of transcripts detected with ≥10 and ≥500 reads were determined from triplicate libraries, with error bars indicating
standard deviation. Salmon v1.5.1 was used for mapping and quantification of all gencode v38 transcripts and ERCCs. (A) Libraries wereprepared from poly(A)-enriched UHRR using the NEBNext UltraExpress RNA Library Prep Kit, Kapa mRNA HyperPrep Kit, orIllumina Stranded mRNA Kit. (B) Libraries were prepared from UHRR using the UltraExpress RNA Library Prep Kit preceded by theNEBNext rRNA Depletion Kit, Kapa HyperPrep Kit with RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero Plus.

The NEBNext UltraExpress RNA Library Prep Kit is the latest generation of NEBNext RNA library prep,with a fast, streamlined workflow featuring:•Fast workflow (3 hours)•Decreased consumable use, fewer steps, shorter incubation times, and fewer cleanups•High-quality directional libraries from a broad input range: 25 - 250 ng total RNA•
Single protocol for all input amounts•
Automation friendly
RNA-seq libraries produced using the The NEBNext UltraExpress RNA Library Prep Kit result in:•High library yields across a range of inputs•
Consistent 5’-3’ transcript coverage•
Greater sensitivity of transcript detection•
Excellent transcript correlation between inputs and replicates and across sample types