A subscription to JoVE is required to view this content. Sign in or start your free trial.
We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2'-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.
The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the 'TruSeq (TS)' protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol 'TS5', which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.
Small RNAs (sRNAs) are involved in the control of a diversity of biological processes1. Eukaryotic regulatory sRNAs are typically between 20 and 30 nt in size; the three major types are microRNAs (miRNA), piwi-interacting RNAs (piRNA) and small interfering RNAs (siRNA). Aberrant miRNA expression levels have been implicated in a variety of diseases2. This underscores the importance of miRNAs in health and disease and the requirement for accurate, quantitative research tools to detect sRNAs in general.
Next-generation sequencing (NGS) is a widely used method to study sRNAs. Main advantages of NGS as compared with other approaches, such as quantitative PCR or microarray techniques (qPCR), are that it does not need a priori knowledge of the sRNA sequences and can therefore be used to discover novel RNAs, and in addition it suffers less of background signal and saturation effects. Further, it can detect single nucleotide differences and has a higher throughput than microarrays. However, NGS also has some drawbacks; the cost of a sequencing run remains relatively high and the multistep process required to convert a sample into a library for sequencing may introduce biases. In a typical sRNA library preparation process, a 3' adapter is first ligated to the sRNA (often gel-purified from total RNA) using a truncated version of RNA ligase 2 (RNL2) and a preadenylated 3' adapter (Figure 1) in the absence of ATP. This increases the efficiency of sRNA-adapter ligation and reduces the formation of side reactions such as sRNA circularization or concatemerization. Subsequently, a 5’ adapter is ligated by RNA ligase 1 (RNL1), followed by reverse transcription (RT) and PCR amplification. All these steps may introduce bias3,4. Consequently, read numbers may not reflect actual sRNA expression levels leading to artificial, method-dependent expression patterns. Specific sRNAs may be either over- or underrepresented in a library, and strongly underrepresented sRNAs may escape detection. The situation is particularly complicated with plant miRNAs, siRNAs in insects and plants, and piRNAs in insects, nematodes and mammals, in which the 3' terminal nucleotide has a 2'-O-methyl (2'-OMe) modification1. This modification strongly inhibits 3' adapter ligation5, making library preparation for these types of RNA a difficult task.
Previous work demonstrated that adapter ligation introduces serious bias, due to RNA sequence/structure effects6,7,8,9,10,11. Steps downstream of adapter ligation such as reverse transcription and PCR do not significantly contribute to bias6,11,12. Ligation bias is likely due to the fact that adapter molecules with a given sequence will interact with sRNA molecules in the reaction mixture to form co-folds, that may either lead to favorable or unfavorable configurations for ligation (Figure 2). Data from Sorefan et al7 suggest that RNL1 prefers a single stranded context, while RNL2 prefers a double strand for ligation. The fact that the adapter/sRNA co-fold structures are determined by the specific adapter and sRNA sequences explains why specific sRNA are over- or underrepresented with a given adapter set. It is also important to note that within a series of sRNA libraries to be compared, the same adapter sequences should be used. Indeed, it has previously been observed that changing adapters by the introduction of different barcode sequences alters miRNA profiles in sequencing libraries9,13.
Randomization of adapter sequences near the ligation junction likely reduces these biases. Sorefan and colleagues7 used adapters with 4 random nucleotides at their extremities, designated "High Definition" (HD) adapters, and showed that the use of these adapters lead to libraries that better reflect true sRNA expression levels. More recent work confirmed these observations and revealed that the randomized region does not need to be adjacent to the ligation junction11. This novel type of adapters was named "MidRand" adapters. Together, these results demonstrate that improved adapter design can reduce bias.
Instead of modifying the adapters, bias can be suppressed through the optimisation of reaction conditions. Polyethylene glycol (PEG), a macromolecular crowding agent known to increase ligation efficiency14, has been shown to significantly reduce bias15,16. Based on these results, several "low bias" kits appeared on the market. These include kits that use PEG in the ligation reactions, either in combination with classical adapters or HD adapters. Other kits avoid ligation altogether, and use 3' polyadenylation and template switching for 3' and 5' adapter addition, respectively12. In yet another strategy, 3' adapter ligation is followed by a circularization step, thus omitting 5' adapter ligation17.
In a previous study, we searched for a sRNA library preparation protocol with the lowest possible levels of bias and the best detection of 2'-OMe RNAs12. We tested some of the above-mentioned 'low bias' kits, which had a better detection of 2'-OMe RNAs than the standard protocol (TS). Surprisingly however, upon modification (the use of randomized adapters, PEG in the ligation reactions and removal of excess 3' adapter by purification) the latter outperformed the other protocols for the detection of 2'-OMe RNAs. Here, we describe in detail a protocol based on the TS protocol, 'TS5', which had the best overall detection of 2'-OMe RNAs. The protocol can be followed using reagents from the TS kit and one reagent from the 'Nf' kit or, to save money, using homemade reagents, with equal performance. We also provide a detailed protocol for the purification of sRNA from total RNA and the preparation of preadenylated 3' adapter.
1. Isolation of small RNAs
2. Preparation of preadenylated 3' HD adapter
NOTE: Preadenylation of 3' HD adapter was done in a manner similar to the protocol described by Chen et al18. Note that preadenylated adapter can be ordered directly (/5rApp/ modification), but this is quite expensive.
3. Library preparation - Protocol TS5
NOTE: We present here the modified TS protocol 'TS5' that we described previously12 and that can be performed either with reagents from the kit or with self-provided reagents. It should be noted that we obtained similar or even slightly better results with a different protocol, 'TS7'. However, with TS7 it is more difficult to eliminate adapter dimers. We have therefore preferred to describe TS5 in detail, but TS7 can be followed by simply replacing the adapters. For TS7 use the 'MidRand-Like (MRL)' adapter sequences (Table 1). Note that here the randomized regions are in the middle of the adapters. Primers for reverse transcription and PCR will hybridize to the sequences downstream of the randomized region in the 3' adapter and upstream of the randomized region in the 5' adapter. Sequencing will start from the first randomized nucleotide in the 5' adapter.
4. Data analysis
NOTE: The data analysis procedure described below is based on the Linux operating system Ubuntu 16.04 LTS.
Critical steps are the isolation of the small RNA fraction of the starting total RNA material (Figure 3) and the desired final library product (Figure 4). Both steps involve polyacrylamide gel purification; small RNA is isolated from 15% TBE urea gels, while the final libraries are isolated from 6% native TBE gels. Small RNA isolated from gel can be analyzed on a small RNA capillary electrophoresis chip (Table of Materials; ...
Small RNA library preparation remains challenging due to bias, mainly introduced during adapter ligation steps. RNAs with a 2'-OMe modification at their 3' end such as plant miRNAs, piRNA in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants are particularly difficult to study because the 2'-OMe modification inhibits 3' adapter ligation. A number of solutions have been proposed in the literature to improve sRNA library preparation protocols, but most commerc...
The authors have nothing to disclose.
This work was supported by the National Center for Scientific Research (CNRS), The French Alternative Energies and Atomic Energy Commission (CEA) and Paris-Sud University. All library preparation, Illumina sequencing and bioinformatics analyses for this study were performed at the I2BC Next-Generation Sequencing (NGS) facility. The members of the I2BC NGS facility are acknowledged for critical reading of the manuscript and helpful suggestions.
Name | Company | Catalog Number | Comments |
2100 Bioanalyzer Instrument | Agilent | G2939BA | |
Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) | ThermoFisher | AM9720 | |
Adenosine 5'-Triphosphate (ATP) | Nex England Biolabs | P0756S | |
Agencourt AMPure XP beads | Beckman Coulter | A63880 | |
Bioanalyzer High Sensitivity DNA Kit | Agilent | 5067-4626 | |
Bioanalyzer Small RNA Kit | Agilent | 5067-1548 | |
Corning Costar Spin-X centrifuge tube filters | Sigma Aldrich | CLS8162-96EA | |
Dark Reader transilluminator | various suppiers | ||
HotStart PCR Kit, with dNTPs | Kapa Biosystems | KK2501 | |
NEXTflex small RNA-seq V3 kit | BIOO Scientific | NOVA-5132-05 | optional |
Novex TBE gels 6% | ThermoFisher | EC6265BOX | |
Novex TBE Urea gels 15% | ThermoFisher | EC6885BOX | |
QIAquick Nucleotide Removal Kit | Qiagen | 28304 | |
Qubit 4 Quantitation Starter Kit | ThermoFisher | Q33227 | |
Qubit ssDNA Assay Kit | ThermoFisher | Q10212 | |
RNA Gel Loading Dye (2X) | ThermoFisher | R0641 | |
RNA Gel Loading Dye (2X) | ThermoFisher | R0641 | |
RNase Inhibitor, Murine | Nex England Biolabs | M0314S | |
SuperScript IV Reverse Transcriptase | ThermoFisher | 18090200 | |
SYBR Gold Nucleic Acid Gel Stain | ThermoFisher | S11494 | |
T4 RNA Ligase 1 (ssRNA Ligase) | Nex England Biolabs | M0204S | |
T4 RNA Ligase 2, truncated | Nex England Biolabs | M0242S | |
TrackIt 50 bp DNA ladder | ThermoFisher | 10488043 | |
TruSeq Small RNA Library Prep Kit | Illumina | RS-200-0012/24/36/48 | optional |
UltraPure Glycogen | ThermoFisher | 10814010 | |
XCell SureLock Mini-Cell | ThermoFisher | EI0001 | |
XCell SureLock Mini-Cell | ThermoFisher | EI0001 | |
ZR small RNA ladder | Zymo Research | R1090 | |
the last two numbers correspond to the set of indexes |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved