5' Capping protocol to add 5’ cap structures to exogenous synthetic RNA references (spike-ins)

doi:10.21203/rs.3.pex-2649/v1

Method Article

5' Capping protocol to add 5’ cap structures to exogenous synthetic RNA references (spike-ins)

https://doi.org/10.21203/rs.3.pex-2649/v1

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Exogenous spike-ins like SIRV and ERCC mixes lack the typical 7-Methylguanosine (m7G) cap structure found in natural eukaryotic RNA transcripts, rendering them incompatible with certain library preparation protocols. This method details the addition of 5’ m7G caps to these spike-ins, enabling their use as external references in cDNA library production dependent on m7G cap structures.

Exogenous synthetic RNA references, known as spike-ins, serve as vital calibration standards in a range of RNA assays, including gene expression microarrays, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and RNA sequencing (RNA-Seq) analyses. Popular spike-ins like Spike-In RNA Variants (SIRV) and External RNA Controls Consortium (ERCC) mixes are widely used due to their reproducibility and well-established concentration levels, enabling accurate quantification and normalization of gene expression across experimental samples^1,2.

However, a significant drawback of commercial spike-in mixes is their lack of the 7-Methylguanosine (m7G) cap structure, typically present at the 5’ end of natural eukaryotic RNA transcripts. This m7G cap plays crucial roles in various cellular processes, including mRNA stability, translation efficiency, and mRNA export from the nucleus. Importantly, many library preparation protocols for RNA sequencing rely on the presence of m7G cap structures to distinguish between complete and incomplete transcripts and to efficiently capture and amplify full-length cDNA molecules.

Due to the absence of m7G caps, commercial spike-ins are unsuitable for library preparation methods requiring 5’ cap enrichment steps, potentially introducing biases and inaccuracies in downstream sequencing analyses. To address this limitation, we present a protocol for modifying synthetic spike-ins by adding 5’ m7G caps, thus enabling their compatibility with library preparation procedures reliant on m7G cap recognition.

In this study, we detail a comprehensive method for introducing 5’ m7G caps to synthetic RNA spike-ins, allowing their use as external references in RNA sequencing experiments. Leveraging the pp5'N structure inherent at the 5’ end of spike-in sequences, we utilize the catalytic activity of the vaccinia capping enzyme to add 7-methylguanylate cap structures^3,4 (Cap 0). Subsequently, RNA magnetic beads are employed to remove protein constituents, including the capping enzyme, from the spike-ins, ensuring their purity and compatibility with downstream library preparation protocols.

This protocol offers a valuable solution for researchers aiming to integrate synthetic spike-ins into RNA sequencing experiments while maintaining the accuracy and fidelity of gene expression measurements.

Here's a categorized list of reagents along with their manufacturers and catalog numbers:

Enzymes and Reagents:

SIRV-Set 4 (Iso Mix E0 / ERCC / Long SIRVs) - Lexogen - 141.0
Agencourt RNAClean XP - BECKMAN COULTER Life Sciences - A63987
RNasin Plus RNase Inhibitor - Promega - N2611
Vaccinia Capping System (10,000 units/ml) - NEW ENGLAND BioLabs - M2080S

Buffers and Solutions:

High Sensitivity RNA sample buffer (Tape Station) - Agilent Technologies - 5067-5580
Ethanol Absolute - Molecular biology grade - PanReac - AppliChem - A8075,1000PE
UltraPure DNase/RNase-Free Distilled Water - INVITROGEN - 10977-035
RNaseZap RNase Decontamination Solution - SIGMA - R2020-250ML

Consumables:

Tube 0.2 ml PCR DNAse/RNAse free - Any brand
Tube 1.5 ml LoBind DNAse/RNAse free - Any brand
Tube 1.5 safe lock DNAse/RNAse free - Any brand
Tube 2 ml DNAse/RNAse free - Any brand
10 µL (0.1 - 10 µL) LongReach Barrier DNAse/RNAse - Any brand
1000 µL (100-1000 µL) Barrier DNAse/RNAse - Any brand
20 µL (1-20 µL) Barrier DNAse/RNAse - Any brand
200 µL (1-200 µL) Barrier DNAse/RNAse - Any brand

Quality Control:

High Sensitivity RNA ScreenTape (Tape Station) - Agilent Technologies - 5067-5579
High Sensitivity RNA ScreenTape Ladder (Tape Station) - Agilent Technologies - 5067-5581
Quanti-it RNA assay kit - Life Technologies - Q32852

Agilent 4200 TapeStation - Agilent Technologies
Qubit 4 Fluorometer - Thermo Fisher Scientific
MJ Research PTC-100 Thermal Cycler - MJ Research
MiniSpin (Centrifuge) - Labnet
MiniSpin (Centrifuge for 8-strip tube strips) - Labnet
IKA MS3 Basic Vortex - IKA
HuLa mixer - Invitrogen
DynaMag-2 (Magnetic Particle Concentrator) - Invitrogen
Thermomixer Compact - Eppendorf
PIPETMAN Classic, P10 - GILSON
PIPETMAN Classic, P1000 - GILSON
PIPETMAN Classic, P2 - GILSON
PIPETMAN Classic, P20 - GILSON
PIPETMAN Classic, P200 - GILSON

Capping reaction

Combine all ten vials of SIRV-Set 4 to achieve a total mass of 535 ng. Ensure the mass falls within the range specified for the NEB Vaccinia capping protocol (max mass = 10 µg). The protocol allows it to be scaled down.
Mix the SIRV-Set 4 vials thoroughly to create a homogeneous 100 µl pool.
Denature the spike-ins by adding 3.5 µl of RNAse inhibitor and 1.5 µl of UltraPure DNase/RNase-Free Distilled Water to the pooled SIRV-Set 4 in a 1.5 ml microfuge tube, reaching a final volume of 105 µl. Incubate the mixture at 65°C for 5 minutes using a thermal block, followed by immediate placement on ice for 5 minutes.
Prepare the capping reaction by sequentially adding the following components and incubate the capping reaction at 37°C for 2 hours:

· 105 µl of denatured spike-ins

· 14 µl of 10X Capping Buffer

· 7 µl of GTP (10 mM)

· 7 µl of SAM (2 mM, dilute 32 mM stock to 2 mM)

· 7 µl of Vaccinia Capping Enzyme

The total volume should reach 140 µl.

Capping reaction purification

After the capping incubation, immediately purify the sample using 1.8x AMPure RNA Clean XP beads.

Incubate the sample for 5 minutes at room temperature using the HuLa mixer.
Quick spin and place the sample on a magnet until the solution clears.
Remove the supernatant.
Wash twice with freshly prepared 70% ethanol solution without disturbing the beads.
Air dry for 1 minute (avoid beads overdrying).
Resuspend the sample in 102 μl of nuclease-free water, and incubate at 37ºC for 5 minutes in a thermo block with gentle rotation.
Place the sample on the magnet until the solution clears, and recover 100 μl of supernatant into a clean tube.

Capping quality control and storage

Quantify the final sample using a Qubit fluorometer and Quanti-it RNA assay kit to check the ratio of SIRV-Set 4 capped vs uncapped.
Analyze the size of capped and uncapped samples of SIRV-Set 4 using TapeStation with the High Sensitivity RNA kit (Figure 1).
Prepare single-use aliquots of capped SIRV-Set 4 and store them at -80ºC for downstream applications.

For the whole procedure, including both the capping reaction and capping reaction purification steps, the approximate time required would be around 3 hours.

Here's a breakdown of the time required for different sections of the procedure:

Capping reaction:

Combining SIRV-Set 4 vials and ensuring the mass falls within the specified range: 10 minutes
Mixing the SIRV-Set 4 vials to create a homogeneous pool: 5 minutes
Denaturing the spike-ins and incubating: 15 minutes
Preparation of the capping reaction components: 5 minutes
Incubation of the capping reaction: 2 hours

Total time for the capping reaction section: Approximately 2 hours and 35 minutes.

Capping reaction purification:

Purification using AMPure RNA Clean XP beads: 20 minutes
Quantification of the final sample using Qubit fluorometer: 10 minutes
Analysis of sample size using TapeStation: 15 minutes
Preparation of single-use aliquots and storage: 5 minutes

Total time for the capping reaction purification section: Approximately 50 minutes.

These times are approximate and may vary based on individual laboratory procedures and equipment availability.

Yang, I. V. Use of external controls in microarray experiments. Methods Enzymol. 411, 50–63 (2006).
Jiang, L. et al. Synthetic spike-in standards for RNA-seq experiments. Genome Res. 21, 1543–51 (2011).
Guo, P. X. & Moss, B. Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 87, 4023–4027 (1990).
Mao, X. & Shuman, S. Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit. Identification of amino acid residues in the D1 protein required for subunit association and methyl group transfer. J. Biol. Chem. 269, 24472–24479 (1994).

We gratefully acknowledge the invaluable assistance and support from the members of the Roderic Guigó lab in the development and optimization of the 5' spike-ins Capping protocol. Their expertise and guidance played a pivotal role in refining the methodology described in this manuscript. We deeply appreciate their contributions to this project.

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5' Capping protocol to add 5’ cap structures to exogenous synthetic RNA references (spike-ins)

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5' Capping protocol to add 5’ cap structures to exogenous synthetic RNA references (spike-ins)

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Abstract

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Introduction

Reagents

Equipment

Procedure

Time Taken

References

Acknowledgements

Associated Publications

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