Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Isolation of small RNAs

1. Extract total RNA using phenol-based reagents or any other method. Verify if the RNA is of good quality.
2. Pre-run a 15% TBE-urea gel (see Table of Materials) for 15 min at 200 V.
3. While the gel is pre-running, mix 5-20 µg of total RNA in a 5-15 µL volume with an equal volume of formamide loading dye (see Table of Materials; 95% deionized formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 5 mM EDTA pH 8) in a 200 µL PCR tube. Likewise, mix 10 µL (200 ng) of small-RNA ladder (see Table of Materials) with an equal volume of formamide load....

Results

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; .......

Discussion

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.......

Materials

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