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., DNA, RNA, proteins, lipids, etc)^5,6,7 of sEVs, once in contact or delivered to recipient cells mediate intercellular communication⁸. Current research efforts have been focused on improving the isolation of cell-specific sEVs from diverse biofluids, to precisely identify and monitor these molecular cargos^9,10. Considering that sEVs contain small non-coding RNAs and particularly microRNAs (miRNAs), which retain their regulatory function upon cellular delivery, many studies have been focused on evaluating the utility of miRNAs as biomarkers that can reveal the condition of their cell of origin^11,12,13.

MicroRNAs (miRNAs) represent a large class (~2,000 known in humans) of small non-coding RNAs with sizes ranging between 19 and 25 nucleotides (nt), which bind to imperfect complementary sites in the 3' untranslated regions of their mRNA targets and direct their degradation and/or post-transcriptional repression^14,15. Functionally, miRNAs have been described to control many biological processes, and the deregulation of their expression has been associated with alterations in molecular, biochemical, and physiological processes that contribute to the initiation and development of diseases, including cancer^16,17,18. Importantly, studies have shown that diseased cells and particularly cancer cells, not only differentially express miRNAs in comparison to normal cells¹⁸, but that their packaging into sEVs also differs^19,20.

While several studies focus on establishing the molecular processes and pathways that direct the packaging of sEVs in normal and diseased cells^19,21,22, biomarker studies are currently focused on identifying distinguishable small non-coding RNA and/or miRNA signatures that are packaged and secreted by specific diseased/cancer cells via sEVs. The targeted isolation of diseased cell-specific sEVs from diverse human biofluids and the evaluation of their multi-omic molecular cargos may thus enable the development of diagnostic and prognostic assays for non-invasive detection of diverse human diseases and cancers^23,24,25,26. Although sEV-miRNAs are the most studied non-coding transcripts, other small non-coding RNA species (i.e., miRNA isoforms (isomiRs), piwiRNAs, transfer RNA fragment (tRFs), rRNAs, orphan non-coding RNAs (oncRNAs)…) are also being evaluated for their potential usefulness as circulating sEV-biomarkers, with specific emphasis on the detection of different cancers^{27,28,29,30,31,32}.

Advantageously, next-generation sequencing (NGS) analysis of small non-coding RNA transcripts (including miRNAs), following their barcoding and cDNA library preparation, is ideal for exploration of known and/or unknown small non-coding RNA species contained in circulating sEVs, which may be associated with diseases²⁷. Indeed, sEV small-RNA NGS provides a high-throughput approach for the discovery of cell-specific small non-coding RNA transcripts that would otherwise remain unknown, if globally evaluated with target-specific technologies (i.e., multiplex PCR, microarrays, customized panels, etc.)^32,33,34. Considering that we previously optimized an ultra-sensitive, highly reproducible cDNA library preparation protocol, which we developed for the analysis of highly degraded and low-concentration formalin-fixed paraffin-embedded (FFPE) RNAs^35,36, we sought to adapt it for the analysis of small non-coding RNAs and miRNAs encapsulated within sEVs^37,38,39,40.

With the discontinuation of the Illumina HiSeq2500 instrument, which provided us with years of high-quality single-end dual index miRNA analysis of sEV cargos^37,40, we sought to explore the reproducibility and sensitivity of our protocol when adapted to updated sequencing instruments using now paired-end dual index chemistry. With the intent to retain the multiplexing capabilities of the original protocol, we maintained the existing 16 barcodes for the preparation of small-RNA cDNA transcripts before integrating them within paired-end dual index chemistry. Using only a pair of i5 and i7 barcodes, we enable the preparation of a robust laboratory-based cDNA library where simultaneous processing and analysis of up to 16 individual samples improve analytical reproducibility of low-input sEV RNA. Thus, we present the biochemical processes, the updated barcodes, PCR and size marker primers, and isolation steps necessary to generate highly reproducible cDNA libraries using ultra-low amounts of miRNAs isolated from human plasma sEVs.

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. Combine the 10 calibrators (10 µL each, Supplemental File 1) into a single 1.5 mL tube to reach a 100 µM stock, dilute it using the carrier oligonucleotide to reach the 0.0052 nM solution, and store at -20 °C.
2. Resuspend each of the 17 unique (16 experimental and 1 test barcoded adapter) adenylated 3' adapters (Figure 1A) with nuclease-free water at a concentration of 50 µM.
   NOTE: Each adapter is aliquoted into individual tubes containing 2.5 µL for storage at -80 °C for up to 2 years, to prevent freeze-thaw cycles of the larger stock.
3. Resuspend the different experimental RNA oligonucleotides, including the RNA extraction carrier (Supplemental File 1), the RNA 3'-ligation carrier (Supplemental File 1), the RNA ligation test (Supplemental File 1), and the 3' ligation (Supplemental File 1; 19 nt-3' adapter and 24 nt-3'adapter) and 5' ligation (Supplemental File 1; 5'adapter-19 nt-3'adapter and 5'adapter-24 nt-3'adapter) DNA size markers, to 250 ng/µL.
4. Dilute the HPLC-purified 5' adapter to 100 µM with nuclease-free water (Figure 1A).
5. Resuspend the RT and first PCR 5' and 3' RT/PCR primers to 100 µM with nuclease-free water (Figure 1B).
6. Resuspend the second PCR 5' and 3' PCR i7 and i5 oligonucleotides to 100 µM with nuclease-free water (Figure 1B).
   NOTE: Aliquot each DNA primer for up to 10 experiments and store at -80 °C.
7. Prepare PolyAcrylamide denAturing (PAA) gel loading dye, mix, and aliquot 1 mL into tubes and store at -80 °C.
   1. Prepare a solution of 0.5 M Na₂H₂ EDTA by adding 18.6 g of Na₂H₂ EDTA to 50 mL of nuclease-free water. Add NaOH pellets slowly to the prepared Na₂H₂ EDTA solution to reach a pH of 8.0. Make up the volume up to 100 mL using ddH₂O to get a 0.5 M Na₂H₂ EDTA (pH 8.0) stock solution
   2. Weigh out 15 mg of Bromophenol Blue into a 15 mL tube, resuspend the powder in 600 µL of nuclease-free water, add 14.25 mL of deionized formamide. Finally, add 150 µL of a 0.5 M Na₂H₂ EDTA, pH 8.0 solution. Aliquot the final PAA solution into 1 mL tubes and store at -80 °C.
   3. Prepare the 5x agarose gel loading dye.
      1. Weigh and dissolve 1.86 g of Na₂H₂-EDTA in 50 mL of RNase-free water and adjust the pH to 8.0 by adding NaOH pellets. Add RNase-free water to 100 mL to get a 50 mM Na₂H₂ EDTA solution.
      2. To 5 mL of 50 mM Na₂H₂ EDTA, add 20 mg of Bromophenol Blue, 20 mg of Xylene Cyanol FF, and 2 g of Ficoll type-400. Vortex the tube containing the three dyes to mix and add an additional 5 mL of 50 mM Na₂H₂ EDTA. Aliquot individual 1 mL tubes of 5x agarose gel loading dye and store at -80°C.

2. Preparation of the EV RNA samples

1. Following the isolation of sEVs, evaluate their morphology using transmission electron microscopy (TEM; Figure 2), concentration using Nanoparticle Tracking Analysis (NTA; Figure 3), and surface protein distribution using super-resolution nanoimaging (Figure 4), or equivalent sEV quantification technology.
2. Aliquot an equivalent number of sEVs in different tubes, add RNase-A (12.5 µg/mL) and incubate at 37 °C for 1 h.
3. Initiate extraction of small non-coding RNAs, including miRNAs, from sEVs using the small-RNA extraction kit and initiate RNA phase isolation.
4. Separate the lower aqueous phase of the RNA lysis buffer, add it to a new tube, and then add 4 ng of RNA carrier oligonucleotide (Supplemental File 1) to each RNA sample, prior to conducting column isolation following the manufacturer's instructions.
5. Speed-vacuum each individual RNA sample to 9.5 µL to initiate the cDNA library preparation protocol.

3. Set up ligations of the 3' barcoded adapter with 16 RNA samples and 1 test sample

1. Identify 16 individual sEV-RNA specimens, aliquot them in 9.5 µL of nuclease-free water into 1.5 mL siliconized microcentrifuge tubes, and place them on ice at least 10 min before starting the experiments.
2. Prepare the 10x RNA Ligase Buffer (without ATP) fresh prior to the experiment.
   1. In a 1.5 mL siliconized tube, combine 343 µL of nuclease-free double-distilled water (ddH₂O), 500 µL of 1 M Tris pH 7.5, 100 µL of 1 M MgCl₂, 50 µL of 20 mg/mL Bovine Serum Albumin, and 7 µL of 14 M 2-mercaptoethanol.
   2. Mix the solution by flicking the tube, centrifuge for 2 s at 2,000 × g at room temperature (RT) on a tabletop centrifuge, and then set on ice.
      NOTE: All steps that describe a "centrifuge for 2 s" are all done with a microcentrifuge at RT with a speed no higher than 2,000 × g.
3. Defrost the 0.0052 nM calibrator cocktail by placing the tube on ice for 10 min.
4. Prepare the Ligation Master Mix for 18 reactions in a 1.5 mL siliconized tube by combining 40 µL of 10x RNA Ligase Buffer, 10 µL of 0.0052 nM calibrator cocktail, and 40 µL of nuclease-free ddH₂O.
5. Flick and spin the 1.5 mL tube and set it on ice.
6. Add 4.5 µL of the Ligation Master Mix to each of the 16 individually aliquoted sEV RNA samples, and the test ligation reaction containing 100 ng of RNA ligation test oligonucleotide (Supplemental File 1) previously aliquoted and at 9.5 µL volume. Flick to mix and spin the tubes before placing them back on ice.
7. Transfer the 16 samples and test reaction to a heat block for 1 min at 90 °C and transfer back to ice.
8. Individually transfer 4 µL of 50% PEG to each tube, flick to mix, and spin the tubes before placing them back on ice.
9. Defrost 2.5 µL aliquots from each of the 17 3' barcoded adapters (16 experimental (#1 to #16) and test adapter (#17)) and place on ice to defrost.
10. Transfer 1 µL of each of the 17 adapters to the corresponding 16 samples containing sEV-RNA and one test sample, with Ligation Master Mix (i.e., 4.5 µL), and 50% PEG (i.e., 4 µL).
    NOTE: All solutions and reactions are mixed by flicking the tubes and spinning them, rather than pipetting the solutions up and down, to prevent loss of material.
11. Prepare the diluted T4 RNA Ligase 2 Truncated K227Q ligation enzyme by adding 10 µL of the enzyme with 10 µL of nuclease-free ddH2O into a new 1.5 mL siliconized tube.
12. Transfer 1 µL of the diluted T4 RNA Ligase 2 Truncated K227Q ligation enzyme into each of the 17 tubes (16 experimental and 1 test RNA samples). Do not pipette up and down, change tips between each tube, keep tubes on ice, flick mix, centrifuge, and place the tubes on ice.
13. Set the ligations onto fresh ice in a tube holder in an ice bucket in the cold room, overnight for up to 18 h.

4. PAGE isolation of the 3' adapter ligated Small RNAs

1. Place the 17 tubes on a heat block at 90 °C for 1 min to deactivate the T4 RNA Ligase 2, and place on ice for 2 min to cool down.
2. Combine 1 µL of GlycoBlue and 26 µL of 5 M nuclease-free NaCl into a fresh tube and transfer 1.2 µL of this Precipitation solution into each of the 17 tubes.
3. Add 63 µL of 100% ethanol to each of the 17 tubes, close, flick, and spin down for 2 s before placing back on ice.
4. Combine the content of the 16 sEV RNA tubes into a single 1.5 mL siliconized tube, while keeping the test RNA ligation tube separate.
5. Invert the tube containing the combined samples 3x to mix, briefly centrifuge for 2 s, and set on ice for 60 min to precipitate.
6. Prepare a large (16 x 20 cm²) 15% polyacrylamide (PAGE) gel as follows:
   1. Siliconize (i.e., using silicone-based solution) and cast the short and long glass plates together with 0.1 cm spacers.
   2. Prepare the 15% PAGE gel mix by combining 9 mL of system diluent, 18 mL system concentrate, 3 mL of system buffer, 240 µL of APS (9%), and 12 µL of TEMED in a 50 mL conical tube.
   3. Quickly transfer the solution between the glass-plates using a 30 mL pipette. Before the gel polymerizes, add a 14-well comb (0.1 cm-thick), and set the gel standing upright for 30 min at RT.
7. Recover the tube with the combined RNA samples from ice and centrifuge it at 16,000 × g for 60 min at 4 °C.
8. Meanwhile, finish setting up the 15% PAGE by gently removing the comb, cleaning the wells with nuclease-free H₂O with a squirt bottle above a sink, before drying and setting up the 15% PAGE on the gel apparatus, where both the upper and lower reservoirs are filled with 0.5x Tris-Borate EDTA (TBE) solution, prior to a 30 min pre-run at 450 V.
9. Remove the tube from the centrifuge (Step 4.6) and carefully dry the RNA pellet using a Pasteur pipette with a 10 µL non-filter tip on its end connected to a vacuum system, without disturbing the pellet.
10. Resuspend the RNA pellet containing the precipitated 16 RNA ligations in 20 µL of nuclease-free ddH₂O and add 20 µL of PAA gel loading solution. Flick the tube to mix, centrifuge for 2 s and set on ice.
11. Replace 0.5x TBE of gel apparatus with fresh 0.5x TBE.
12. Set up a tube with 20 bp RNA size ladder, two tubes with each of the two 3'-ligation short DNA size markers (Supplemental File 1), and one tube containing the RNA ligation test oligonucleotide (i.e., ligated with Barcoded adapter #17).
13. To each of the tubes (i.e., ladder, 3' ligation short DNA oligos, and RNA ligation test), add 20 µL of PAA gel loading solution.
14. Set the 16 ligated RNA sample tube and 1 RNA ligation test tube at 90 °C for 1 min and set back on ice.
15. Replace 0.5x TBE buffer from both lower and upper reservoirs (i.e., add buffer below the level of well so there is no cross-contamination between wells during loading) and load the ladder on the ends of the gel, the RNA test ligation, the two short DNA size markers on both sides of the well containing the 16 combined 3' barcoded RNA ligations (see Figure 5).
16. Run the 15% PAGE gel with all samples for 90 min at 450 V (~35 mA) under cooled conditions (i.e., with fans blowing air on the glass plates).
17. Remove 15% PAGE from glass and lightly spray it with SYBR Gold solution (10 µL SYBR Gold in 25 mL of 0.5x TBE) and let it sit tilted for 5 min in the dark.
18. Set the gel on a blue-light transilluminator (Figure 5) and align both 19 nt-3' adapter and both 24 nt- 3' adapter size marker short DNA oligonucleotides with a ruler and excise the area of the gel containing the ligated miRNA constructs (Figure 6 displays the 3' ligation and resulting construct). Separately, excise the upper band of the RNA test ligation, which will also be used for a test 5' ligation the following day.
19. Place the two separate excised gel pieces (i.e., for 16 RNA samples and for test ligation) into different 0.5 mL gel breaker tubes set into 1.5 mL siliconized tubes, centrifuge at 16,000 × g for 3 min at RT and then resuspend the two set fragmented gel pieces with 300 µL of 400 mM NaCl solution, close the tubes, and seal them with parafilm.
20. Set the tubes on a shaker with agitation at 1,100 rpm at 4 °C, overnight (16-17 h).

5. Ligation of the 5' adapter

1. Transfer two separate fragmented gels and their solutions (16 x 3' barcoded samples (barcodes #1 to #16) and 3' barcoded test RNA (barcode #17)) onto two separates 5 µm filter tubes, each set into 1.5 mL siliconized tubes, seal them with parafilm, and centrifuge for 5 min at 2,300 × g at RT.
2. Add 950 µL of 100% ethanol to each filtered solution, close the tubes, invert to mix, spin down for 2 s, seal the tubes with parafilm, and set them on ice for 1 h.
3. Freshly prepare a 50% aqueous DMSO stock by combining 1 mL of nuclease-free water with 1 mL of DMSO, wrap the tube in aluminum foil, and store at RT in the dark.
4. Prepare a 15% PAGE gel and pre-run it, following the steps described in section 4.6.
5. Centrifuge the tubes containing the 3' ligated 16 RNA samples and the test RNA ligation at 16,000 × g for 60 min at 4 °C to precipitate the ligated RNAs.
6. Carefully remove the supernatant without touching the RNA pellets, and vacuum dry.
7. Resuspend the 3' ligated pellet of 16 RNA samples and test RNA each in 8 µL of nuclease-free ddH₂O without pipetting up and down. Lightly flick the tubes to mix, centrifuge for 2 s (2,000 × g) at RT, and set the tubes on ice.
8. Prepare the 10x RNA Ligase Buffer (with ATP) by mixing 500 µL of 1 M Tris (pH 7.5), 100 µL of 1 M MgCl₂, 50 µL of 20 mg/mL acetylated BSA, 200 µL of 10 mM ATP, 7 µL of 2-mercaptoethanol 14 M, and 143 µL of RNase-free water into a 1.5 mL tube.
9. Add 2 µL of 10x RNA ligase buffer (i.e., with ATP), 1 µL of 100 µM 5' adapter, and 4 µL of 50% aqueous DMSO to each tube. Flick to mix, spin for 2 s at RT, then set up the tube at 90 °C for 1 min to denature the RNA, and place it back on ice.
10. Add 3 µL of 50% DMSO, flick to mix, spin to pellet, add 3 µL of T4 RNA Ligase to each tube, flick the tubes, centrifuge for 2 s at RT, and set the tubes on the shaker at 37 °C for 75 min.
    NOTE: Do not pipette up and down.
11. Prepare the 20 nt DNA ladder and two separate tubes containing the 5'-ligation size marker long DNA oligonucleotides (Supplemental File 1; 5'adapter-19 nt-3' adapter, and 5'adapter-24 nt-3'adapter), and add 20 µL of PAA to each of the tubes.
12. Once the 5' adapter ligation is complete, add 20 µL of PAA to each of the two ligations (i.e., 16 RNA samples and test RNA), mix by flicking, spin down, and set at 90 °C for 1 min, after which place the tubes back onto ice.
13. Empty upper and lower reservoirs of gel apparatus and add fresh 0.5x TBE solution below the level of the wells of the upper reservoir.
14. Load the ladder on the gel's end, the 5' ligated test RNA sample, the two size-marker long DNA oligonucleotides (i.e., 5' adapter-19 nt-3' adapter, and 5' adapter-24 nt-3' adapter) in wells surrounding the well where the 5' ligated 16 RNA samples will be loaded (Figure 6 displays the 5' ligation and resulting construct). Run the gel for 90 min at 450 volts.
15. Once the run is complete, remove the gel from the apparatus and glass and spray it with a SYBR Gold solution (i.e., 10 µL of SYBR Gold in 25 mL of 0.5x TBE), and let it sit in the dark for 5 min.
16. Using the 5' adapter-19 nt-3'adapter and both 5'- adapter-24 nt-3' adapter size markers long DNA oligonucleotides as size guides, directly excise the section of the gel containing the 5' ligated 16 RNA samples (Figure 7).
    NOTE: The test RNA ligation is evaluated on the gel to demonstrate that the ligation of the 5' adapter allows a shift equivalent in size to the size markers. The test RNA ligation is not excised from the gel; only the ligated 16 combined RNA samples.
17. Transfer the excised gel piece containing the 16 ligated RNA samples into a 0.5 mL gel breaker tube set into a 1.5 mL tube, spin at 16,000 × g for 3 min at RT, and then add 300 µL of 300 mM NaCl and 1 µL of 100 µM 3' RT/PCR primer (Figure 1B) to the crushed gel pieces in the tube.
18. Seal the tube with parafilm and set on a shaker agitating at 1,100 rpm at 4 °C, overnight (17-18 h) in a cold room.

6. Reverse transcription of the barcoded 5' and 3'-ligated small non-coding RNAs and miRNAs

1. Pipette the solution from the tube containing the crushed gel onto a 5 µm filter tube inserted into a 1.5 mL siliconized RNase-free tube and briefly spin to allow the solution to get through the filter.
2. Add 950 µL of 100% ethanol to this filtered solution, invert the tube to mix, spin for 2 s, and set on ice for 60 min.
3. Precipitate the RNA pellet by centrifugation of the tube at 16,000 × g at 4 °C for 1 h.
4. Recover the tube, remove the supernatant carefully without touching the pellet, and vacuum dry the pellet prior to resuspending it in 5.6 µL of nuclease-free H₂O.
5. Set up the RT reaction by adding 3 µL of 5x first strand buffer, 4.2 µL of 10x dNTPs (each at 2 mM), and 1.5 µL of Dithiothreitol (DTT) to the tube. Flick to mix, and spin for 2 s.
6. Set up the reaction at 90 °C for 30 s exactly and then transfer the tube to a block at 50 °C for 2 min.
7. Initiate the RT reaction by adding 0.75 µL of reverse transcriptase enzyme into the solution, flick to mix, and set at 50 °C for 35 min.
8. Transfer the tube at 95 °C for 1 min to denature the enzyme and the cDNA/RNA strands, then add 95 µL of RNase-free H₂O, flick the tube to mix, and set it on ice for 2 min. This tube contains cDNA generated from the 16 RNA samples, each separately barcoded in 3'.

7. Conduct the test PCR and large-scale amplification

1. Prepare fresh 10x PCR buffer by combining 304 µL of Nuclease-free H₂O, 125 µL of 2 M KCl, 50 µL of 1 M Tris pH 8.0, 10 µL of 1 M MgCl₂, 5 µL of 1% Triton X-100, and 6 µL of 1 M 2-mercaptoethanol into a tube that can be stored on ice.
2. Assemble a Pilot/test PCR reaction for the cDNA produced in step 6.8 (above) by combining 67 µL of Nuclease-free H₂O with 10 µL of 10x PCR Buffer, 10 µL of 10x dNTPs, 0.5 µL of 100 µM 5' PCR primer, 0.5 µL of 100 µM 3' PCR primer, 10 µL of cDNA Stock Library, and 2 µL of 50x Taq polymerase into a fresh PCR tube.
   NOTE: Prior to conducting the PCR amplifications, set up the instrument by establishing two different files as follows: For File#1: 94 °C for 45 s, 50 °C for 85 s, and 72 °C for 60 s for 12 cycles, and 4 °C; and for File#2: 94 °C for 45 s, 50 °C for 85 s, and 72 °C for 60 s for 2 cycles, and 4 °C.
3. For the pilot/test PCR reaction, place the PCR tube containing the 100 µL of the PCR reaction in the Thermocycler and run File#1.
4. Once File#1 is complete, open the PCR tube and transfer 12 µL of the reaction into a 1.5 mL microcentrifuge containing 3 µL of 5x gel loading dye. Close the tube, and write "12 cycles" on the cap.
5. Take the PCR tube, set it into the thermocycler and initiate File#2.
6. Once File#2 is complete, transfer 12 µL of the PCR reaction into a 1.5 mL tube containing 3 µL of 5x gel Loading dye, and write "14 cycles" on its cap.
7. Repeat the PCR amplifications four successive more times (i.e., two PCR cycles each), while transferring 12 µL of PCR product at the end of each amplification, and in turn obtain four different tubes, labelled 16, 18, 20, and 22 cycles.
8. Load the 20 nt size ladder, and each of the PCR amplified products onto a 2.5% agarose and run the gel in 0.5x TBE for 30 min at 120 V.
9. Identify the optimal PCR amplification cycle (Figure 8A), based on the ratio and presence of primer dimers (lower band) and the amplified cDNA (upper band).
   NOTE Considering that the total RNA input with 16 samples may be lower than 1 ng, it is expected that primer dimers will generate a product quicker and in larger quantities than the purified cDNA. Thus, the adequate PCR cycle is generally selected between 18 and 22 cycles. In Figure 8A, the adequate PCR cycle for this library is 20. In Figure 8B, the size of the PCR construct is detailed.
10. Once the adequate PCR amplification cycle is identified, set a negative PCR (no cDNA) and four 100 µL positive PCR reactions (i.e., each containing 12 µL of cDNA).
    1. To simplify the preparation of the PCR reactions, set up a PCR Master Mix (i.e., for 4.5 reactions) in a 1.5 mL tube by combining 292.5 µL of nuclease-Free H₂O, 45 µL of 10x PCR buffer, 45 µL of 10x dNTPs, 3.25 µL of 3' RT/PCR primer (Figure 1B) and 2.25 µL of 5' PCR primer (Figure 1B). Invert this tube to mix and spin to collect at the bottom of the tube.
    2. Transfer 86 µL of the PCR Master Mix into each of four 0.5 mL PCR tubes.
    3. Add 12 µL of cDNA Library (stored on ice), then 2 µL of 50x titanium Taq polymerase into each of the 4 PCR tubes, and mix by pipetting up and down.
    4. Prepare the no-template PCR reaction tube by adding 77 µL of nuclease-free H₂O, 10 µL of 10x PCR buffer, 10 µL of 10x dNTPs, 0.5 µL of 5' PCR primer, 0.5 µL of 3' PCR primer, and 2 µL of 50x Taq polymerase to a separate PCR tube and pipetting up and down to mix.
    5. Set up the PCR tubes in the thermocycler using the PCR cycle identified in step 7.9 and set a new program on the thermocycler to amplify as necessary.
11. Prepare a 2.5% agarose gel with 0.5x TBE and ethidium bromide, and transfer 12 µL from each PCR tube into individual tubes containing 3 µL of 5x gel loading dye.
12. Prepare the 20 nt ladder by combining 3 µL of ladder with 9 µL of nuclease-free H₂O and 3 µL of gel loading dye.
13. Load the gel, run it for 30 min at 120 V, and verify that the PCR amplification is similar between the four different reactions and that the no-template control is empty (Figure 8C).
14. Combine the four PCR reactions between two 1.5 mL siliconized microcentrifuge tubes (i.e., two tubes each containing 90 µL of product), and add 18 µL of 5 M NaCl, and 800 µL of 100% ethanol in each of the two tubes.
    1. Invert the tubes to mix and store them at -20 °C to precipitate overnight.

8. Purification of the PCR amplified Library

1. Centrifuge the two tubes containing the mixed positive PCR reactions at 16,000 × g for 60 min at 4 °C.
2. After 60 min, remove the supernatant, vacuum dry the DNA pellets, and resuspend them in 19.5 µL of 1x buffer, and then add 0.5 µL of PmeI enzyme.
3. Set up the PmeI digests at 37 °C for 2 h to remove any of the DNA size markers that ran in the adjacent wells (i.e., 19 nt-3' adapter, 24 nt-3'adapter) or RNA carriers (i.e., RNA extraction carrier, RNA post-3' ligation carrier) potential contaminants (i.e., see PmeI sites included in RNA and DNA primers in Supplemental File 1)
4. Run the two digests in three different wells of a 2.5% agarose gel using 0.5x TBE to avoid accumulating an excess of primer dimers in a single well and creating a smear.
   1. Add 3 µL of 5x gel loading dye to each of the digests, combine the two digests, and then load 16.5 µL into three separate adjacent wells.
   2. Load the 20 nt DNA size ladder at the end wells of the gel and run the gel for 90 min at 150 V (Figure 8D).
5. To purify the DNA libraries, excise the upper PCR bands from the three adjacent lanes of the gel, and transfer them into a fresh 1.5 mL tube.
6. Purify the PCR amplified library using a gel extraction kit, following the manufacturer's instructions, and quantify the purified DNA product using a fluorometer, following manufacturer's instructions.

9. Final PCR amplification to add barcodes compatible with the sequencer

1. Prepare a fresh 10x PCR buffer mix as the one described in step 7.1.
2. Set up PCR reactions by including one negative control (no amplicons) and three PCR reactions using 0.75 ng of the PCR product purified from the first PCR amplification in each of the three tubes as follows:
   1. Set up three individual PCR reactions by mixing up 75 µL of ddH₂O, 10 µL of 10x PCR buffer, 10 µL of 10x dNTPs, 1 µL of 5' 2^nd PCR primer (i.e., primer i503 (Figure 1B)), 1 µL of 3' PCR 2^nd primer (i.e., primer i702 (Figure 1B)), 1 µL of PCR product (i.e., 0.75 ng from gel-purified 1^st PCR product), and 2 µL of Titanium Taq Polymerase.
   2. Set up the negative PCR control (i.e., no DNA template) by mixing up 76 µL of ddH₂O, 10 µL of 10x PCR buffer, 10 µL of 10x dNTPs, 1 µL of 5' 2^nd PCR primer (i.e., primer i503 (Figure 1B)), 1 µL of 3' PCR 2^nd primer (i.e., primer i702 (Figure 1B)), and 2 µL of Taq Polymerase.
   3. Set up the four PCR reactions (i.e., one negative control, three positives) on a thermocycler programmed as follows: 72 °C for 3 min, 95 °C for 30 s, and 95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s for 9 cycles, and then 72 °C for 5 min, and a final hold at 10 °C.
3. Once the PCR cycles are completed, take 10 µL of the reaction mixture and add 3 µL of loading dye, and evaluate the products on a 2.5% agarose gel as displayed in Figure 9A, along with the 20 nt ladder.
4. Upon validation of the PCR product's size (i.e., upper PCR band running at 164 bp as displayed in Figure 9B and Figure 6 for size and details on the final construct) and band distribution, combine the PCR products in two tubes by adding to each 135 µL of PCR product (i.e., 90 µL+ 45 µL), 13.5 µL of 5 M NaCl, and 650 µL of 100% ethanol, and precipitate at -20 °C overnight.
5. The next day, spin down the two PCR tubes at 16,000 × g for 60 min at 4 °C.
6. Dry each pellet, resuspend with 19.5 µL of 1x PmeI buffer, and add 0.5 µL of PmeI for 2 h at 37 °C on a shaker to further eliminate any of the size markers that ran in the adjacent wells (i.e., 19 nt-3' adapter, 24 nt-3'adapter) or RNA carriers (i.e., RNA extraction carrier, RNA post-3' ligation carrier) contaminants.
7. Set up the digested products in three wells of a 2.5% agarose gel and separate at 150 V for 90 min.
8. Excise the upper band, which runs at 164 bp (Figure 9C), and purify the DNA product using a DNA isolation kit and quantify with a fluorometer.
9. Run the final PCR product on a DNA chip to validate size, concentration, and purity of the library before dilutions and analysis using a sequencer.
10. Transfer the FASTQ data file to the RNAworld pipeline for adapter trimming, demultiplexing, alignment to the human genome, prior to miRNA analyses, as previously described⁴¹. Alternatively, using the Script presented in Supplemental File 3, demultiplex the reads from the FASTQ file and further process using a small-RNA analytical pipeline of choice.

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