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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 NG....
1. Isolation of small RNAs
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.......
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 |
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