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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

AQRNA-seq for Quantifying Small RNAs

Published: February 2nd, 2024

DOI:

10.3791/66335

1Department of Biological Engineering, Massachusetts Institute of Technology, 2Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

Next-generation sequencing (NGS), also known as massively parallel sequencing, is a DNA sequencing technology that involves DNA fragmentation, ligation of adaptor oligonucleotides, polymerase chain reaction (PCR)-based amplification, sequencing of the DNA, and reassembly of the fragment sequences into a genome. The adaptation of NGS to sequence RNA (RNA-seq) is a powerful approach to identify and quantify RNA transcripts and their variants1. Innovative developments in RNA library preparation workflows and bioinformatic analysis pipelines, coupled with advancements in laboratory instrumentation, have expanded the repertoire of RNA-seq applicatio....

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NOTE: Figure 1 provides a graphical illustration of the procedures involved in AQRNA-seq library preparation. Detailed information regarding the reagents, chemicals, and columns/kits used in the procedure can be found in the Table of Materials. It is recommended to perform a comprehensive evaluation of purity, integrity, and quantity of the input RNA samples using (i) 3% agarose gel electrophoresis, (ii) automated electrophoresis tools for sample quality control of biomolecu.......

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Mycobacterium bovis BCG (bacilli de Calmette et Guérin) strain 1173P2 undergoing exponential growth were subject to a time series (0, 4, 10, and 20 days) of nutrient starvation, followed by a 6-day resuscitation in nutrient-rich medium as previously presented in Hu et al.7. Small RNAs were isolated from bacterial culture, with three biological replicates, at each of the five designated time points. Illumina libraries were constructed using the above-described AQRNA-seq library prepar.......

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The AQRNA-seq library preparation workflow is designed to maximize the capture of RNAs within a sample and minimize polymerase fall-off during reverse transcription7. Through a two-step linker ligation, novel DNA oligos (Linker 1 and Linker 2) are ligated in excess to fully complement the RNA within the sample. Excess linkers can be efficiently removed with RecJf, a 5' to 3' exonuclease specific to single-stranded DNAs, leaving the ligated products intact. In addition, AlkB treatment reduc.......

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The authors of the present work are grateful to the authors of the original paper describing the AQRNA-seq technology7. This work was supported by grants from the National Institutes of Health (ES002109, AG063341, ES031576, ES031529, ES026856) and the National Research Foundation of Singapore through the Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance IRG.

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NameCompanyCatalog NumberComments
2-ketoglutarate Sigma-Aldrich75890Prepare a working solution (1 M) and store it at -20ºC
2100 Bioanalyzer InstrumentAgilentG2938C
5'-deadenylase (50 U/μL) New England BiolabsM0331S (component #: M0331SVIAL)Store at -20 °C
Adenosine 5'-Triphosphate (ATP) New England BiolabsM0437M (component #: N0437AVIAL)NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM); prepare a working solution (10 mM) and store it at -20ºC
AGAROSE GPG/LEAmericanBioAB00972-00500Store at ambient temperature
Ammonium iron(II) sulfate hexahydrateSigma-AldrichF2262Prepare a working solution (0.25 M) and store it at -20 °C
Bioanalyzer Small RNA AnalysisAgilent5067-1548 The Small RNA Analysis is used for checking the quality of input RNAs and the efficiency of enzymatic reactions (e.g., Linker 1 ligation)
Bovine Serum Albumin (BSA; 10 mg/mL) New England BiolabsB9000This product was discontinued on 12/15/2022 and is replaced with Recombinant Albumin, Molecular Biology Grade (NEB B9200).
Chloroform Macron Fine Chemicals4441-10
Demethylase ArrayStarAS-FS-004Demethylase comes with the rtStar tRNA Pretreatment & First-Strand cDNA Synthesis Kit (AS-FS-004)
Deoxynucleotide (dNTP) Solution MixNew England BiolabsN0447L (component #: N0447LVIAL)This dNTP Solution Mix contains equimolar concentrations of dATP, dCTP, dGTP and dTTP (10 mM each)
Digital Dual Heat BlockVWR Scientific Products13259-052Heating block is used with the QIAquick Gel Extraction Kit 
DyeEx 2.0 Spin Kit Qiagen63204 Effective at removing short remnants (e.g., oligos less than 10 bp in length)
Electrophoresis Power SupplyBio-Rad LabrotoriesPowerPac 300
Eppendorf PCR Tubes (0.5 mL)Eppendorf0030124537
Eppendorf Safe-Lock Tubes (0.5 mL)Eppendorf022363611
Eppendorf Safe-Lock Tubes (1.5 mL)Eppendorf022363204
Eppendorf Safe-Lock Tubes (2 mL)Eppendorf022363352
Ethyl alcohol (Ethanol), PureSigma-AldrichE7023 The pure ethanol is used with the Oligo Clean and Concentrator Kit from Zymo Research
Gel Imaging SystemAlpha InnotechFluorChem 8900
Gel Loading Dye, Purple (6X), no SDSNew England BiolabsN0556S (component #: B7025SVIAL)NEB N0556S contains Quick-Load Purple 50 bp DNA Ladder and Gel Loading Dye, Purple (6X), no SDS
GENESYS 180 UV-Vis SpectrophotometerThermo Fisher Scientific840-309000The spectrophotometer is used for measuring the oligo concentrations using the Beer's law
HEPESSigma-AldrichH4034Prepare a working solution (1 M; pH = 8 with NaOH) and store it at -20 °C
Hydrochloric acid (HCl) VWR Scientific ProductsBDH3028 Prepare a working solution (5 M) and store it at ambient temperature
Isopropyl Alcohol (Isopropanol), PureMacron Fine Chemicals3032-16Isopropanol is used with the QIAquick Gel Extraction Kit 
L-Ascorbic acidSigma-AldrichA5960Prepare a working solution (0.5 M) and store it at -20ºC
MicrocentrifugeEppendorf5415D
NanoDrop 2000 SpectrophotometerThermo Fisher ScientificND-2000
NEBuffer 2 (10X)New England BiolabsM0264L (component #: B7002SVIAL)NEB M0264L contains RecJf (30 U/μL) and NEBuffer 2 (10X); store at -20 °C
Nuclease-Free Water (not DEPC-Treated)Thermo Fisher ScientificAM9938 
Oligo Clean & Concentrator Kit Zymo ResearchD4061 Store at ambient temperature
PEG 8000 (50% solution) New England BiolabsM0437M (component #: B1004SVIAL)NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM); prepare a working solution (10 mM) and store it at -20ºC
Peltier Thermal CyclerMJ ResearchPTC-200
Phenol:choloroform:isoamyl alcohol 25:24:1 pH = 5.2 Thermo Fisher ScientificJ62336 
PrimeScript Buffer (5X)TaKaRa2680A 
PrimeScript Reverse TranscriptaseTaKaRa2680A 
QIAquick Gel Extraction Kit Qiagen28704This kit requires a heating block and isopropanol to work with
Quick-Load Purple 100 bp DNA LadderNew England BiolabsN0551S (component #: N0551SVIAL)
Quick-Load Purple 50 bp DNA LadderNew England BiolabsN0556S (component #: N0556SVIAL)NEB N0556S contains Quick-Load Purple 50 bp DNA Ladder and Gel Loading Dye, Purple (6X), no SDS
RecJf (30 U/μL) New England BiolabsM0264L (component #: M0264LVIAL)NEB M0264L contains RecJf (30 U/μL) and NEBuffer 2 (10X); store at -20 °C
RNase Inhibitor (murine; 40 U/μL) New England BiolabsM0314L (component #: M0314LVIAL)Store at -20 °C
SeqAMP DNA Polymerase TaKaRa638509 TaKaRa 638509 contains SeqAMP DNA Polymerase and SeqAMP PCR Buffer (2X)
SeqAMP PCR Buffer (2X) TaKaRa638509 TaKaRa 638509 contains SeqAMP DNA Polymerase and SeqAMP PCR Buffer (2X)
Shrimp Alkaline Phosphatase (1 U/μL) New England BiolabsM0371L (component #: M0371LVIAL)
Sodium hydroxide (NaOH)Sigma-Aldrich S5881Prepare a working solution (5 M) and store it at ambient temperature
T4 DNA Ligase (400 U/μL) New England BiolabsM0202L (component #: M0202LVIAL)NEB M0202L contains T4 DNA Ligase (400 U/μL) and T4 DNA Ligase Reaction Buffer (10X)
T4 DNA Ligase Reaction Buffer (10X) New England BiolabsM0202L (component #: B0202SVIAL)NEB M0202L contains T4 DNA Ligase (400 U/μL) and T4 DNA Ligase Reaction Buffer (10X)
T4 RNA Ligase 1 (30 U/μL) New England BiolabsM0437M (component #: M0437MVIAL)NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM)
T4 RNA Ligase Reaction Buffer (10X) New England BiolabsM0437M (component #: B0216SVIAL)NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM)

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