Ultrasensitive cDNA Library Preparation for Next-generation Sequencing of MicroRNAs from Small Extracellular Vesicles

Summary

Extracellular vesicles (EVs) contain cell-specific microRNAs that regulate recipient cells, the identification of which may shed light on their function and role as biomarkers. Our optimized cDNA library preparation protocol introduces unique barcodes that enable sample multiplexing and enhanced processing of low-input EVs while using paired-end dual index barcodes for compatibility with Illumina sequencers.

Abstract

Recent studies demonstrate that small extracellular vesicles (sEVs), which are found in all biofluids, play critical roles in intercellular communication by channeling proteins, DNA, and RNAs. MicroRNAs (miRNAs) that are packaged in sEVs have emerged as critical deliverable regulators in recipient cells. Since sEVs secreted by normal and diseased cells carry different miRNA cargos, recent sEV-miRNA profiling studies suggest that they may help identify novel circulating biomarkers. However, cell/disease-specific sEVs circulating in diverse biofluids, once isolated, provide low miRNA quantities, which are generally difficult to quantify using conventional spectrometric methodologies. Small non-coding RNA Next Generation Sequencing (NGS), which allows for the amplification of cloned miRNA sequences, offers a valuable opportunity to evaluate the miRNA cargos of sEVs. Unfortunately, commercial cDNA library preparation procedures often require RNA inputs well above the unquantifiable amounts available from isolated sEVs.

Thus, considering the robustness and multiplexing capabilities of our existing cDNA library preparation procedure (i.e., initially optimized for the analysis of low-input, highly degraded, formalin-fixed paraffin-embedded (FFPE) RNA), we sought to evaluate its applicability for the analysis of sEV miRNAs. Importantly, taking into account the recent technical clustering improvements of sequencing chips, we sought to adapt our transcript barcoding approach within a paired-end, dual index-compatible cDNA library preparation workflow to enhance our sequencing and multiplexing capabilities. Using RNA extracted from 8.4 × 10⁹ sEVs in 16 replicates, and from decreasing amounts of sEVs from 10¹⁰ sEVs to as low as 2.5 × 10⁷ sEVs, we evaluated the reproducibility and sensitivity of this methodology. The data demonstrate that the 16 3' adenylated DNA barcodes allow for highly reproducible and sensitive detection of sEV-miRNA profiles across repeats using as low as 3.15 pg of total small non-coding RNAs or 1.35 pg of miRNAs.

Introduction

Small extracellular vesicles (sEVs) are nanosized (~30-200 nm in diameter) cell-derived particles enveloped by a phospholipid bilayer membrane, which is inherited from their cell of origin and that robustly protects their molecular cargos^1,2. It is well accepted that virtually all cells produce and release sEVs into the intercellular compartment, which can in turn be detected in most biofluids (i.e., blood, urine, saliva, etc)^2,3,4. Recent studies have demonstrated that the stably encapsulated molecular cargos (i.e.,&....

Protocol

1. All reagents and oligonucleotides are prepared as follows:

NOTE: All nucleotides used in this protocol are detailed in Figure 1 and Supplemental File 1 at concentrations that are used for implementing this protocol. See Supplemental File 2 for primer sequences.

1. Prepare a stock solution of calibrator cocktail, which will be used in each individual ligation as a control for the different enzymatic reactions:
   1. Resuspend the Carrier oligonucleotide (0.5 µM) (Supplemental File 1) with RNase-free water.
   2. Combi....

Results

As detailed in the above protocol, we describe the simultaneous processing of up to 16 individual small extracellular vesicle (sEV) RNA samples (i.e., for analysis of small non-coding RNAs and miRNAs), which are analyzed together within a single library, after undergoing 3' barcoding. The sEVs that were selected for our analyses were isolated by ultracentrifugation, as previously described^37,40, from 45 mL of plasma collected from women diagnosed with triple nega.......

Discussion

We have developed a cDNA library preparation protocol for reproducible and sensitive next-generation sequencing (NGS) of non-coding RNAs, including microRNAs (miRNAs) efficiently isolated from small extracellular vesicles (sEVs). Considering that a limited quantity of small non-coding RNAs and miRNAs may be recovered from globally or selectively isolated sEVs, we sought to optimize our cDNA library preparation procedure that enables reproducible and sensitive analysis of sEVs miRNA cargos.

As .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% Triton X-100	Invitrogen	HFH10
10 mM ATP	Ambion	AM8110G
10x dNTPs	Ambion	AM8110G
10x TBE	Thermofisher Scientific	15581044
14 M Mercaptoethanol	Sigma	O3445I-100
20 nt ladder	Jena Bioscience	M-232S
20 mg/mL Bovine Serum Albumin	Sigma	B8894-5ML
50x Titanium Taq	Clontech Laboratories	639208
Ammonium Persulfate	Fisher Scientific	7727-54-0
Blue light transilluminator- Safe Imager 2.0	Thermofisher	G6600
BRL Vertical Gel Electrophoresis System with glass plates and combs	GIBCO	V16
Dimethyl sulfoxide (DMSO)	Sigma	D9170-5VL
Eppendorf microcentrifuge 5424R	USA scientific	4054-4537Q
Eppendorf Thermomixer	USA scientific	4053-8223Q
Filter tube with 5mm filter	IST Engineering Inc.	5388-50
Fisherbrand Siliconized Low-Retention Microcentrifuge Tubes 1.5 mL	Fisher Scientific	02-681-320
Gel Breaker Tube 0.5 mL	IST Engineering Inc.	3388-100
Gel electrophoresis apparatus 7 cm x 10 cm- Mini-sub Cell GT with gel trays and combs	Biorad	1704446
Glycoblue	Ambion	AM9516
Illumina NextSeq 1000/2000 sequencer	Illumina	20047256
Jersey-Cote (Silicon-based solution)	LabScientific, Inc	1188
KCl 2 M	Ambion	AM9640G
MgCl2 1 M	Ambion	AM9530G
Minifuge dual rotor personal centrifuge	USA scientific	2641-0016
Model V16 polyacrylamide gel electrophoresis apparatus, glasses, combs, and spacers	Ciore Life Science	21070010
Oligonucleotides	IDT	Defined during order
Owl EasyCast B2 mini electrophoresis system- with gel trays and combs	Thermofisher Scientific	B2
Qiaquick Gel Extraction kit	Qiagen	28704
Qubit Fluorometer	Thermofisher Scientific	Q33238
Restriction enzyme PmeI and 10x Cut Smart Buffer	NEB	R0560S
Reverse transcriptase- Superscript III	ThermoFisher	12574026
RNase-free water	Ambion	AM9932
Shaker- Eppendorf Thermomixer	Eppendorf	5385000024
SeaKem LE agarose	Lonza	50002
Superscript III reverse transcription kit	Invitrogen	18080-044
SYBR Gold	Life Technologies	S11494
Titanium Taq Polymerase	Takara	639208
T4 RNA Ligase 1	NEB	M0204S
T4 RNA Ligase 2 Truncated K227Q	NEB	0351L
TEMED	Fisher Scientific	O3446I-100
Themocycler with heated lid	Applied Biosystem	4359659
Tris 1 M pH 7.5	Invitrogen	15567027
Tris 1 M pH 8.0	Ambion	AM9855G
UltraPure Sequagel system concentrate, diluent, and buffer	National Diagnostics	EC-833