Direct RNA Sequencing (RNA004) Protocol

Information

This protocol is explicitly for the new SQK-RNA004 Nanopore kit. If you are using the discontinued SQK-RNA002 kit and either an R9.4.1 flow cell or flongle, please see the old Nanopore RNA Sequencing Protocol (SQK-RNA002).

This protocol is a modified version of the Nanopore SQK-RNA004 protocol available here, and the first part series of my m6A Detection Protocol. This protocol will cover the library preparation portion and beginning of the sequencing run, while the m6A detection will be handled in a separate article.

Introduction

This protocol is designed to guide you through the preparation of an RNA library for sequencing through nanopore, adapted from the Oxford Nanopore Technologies Direct RNA Sequencing SQK-RNA004 protocol. Following the sequencing run, you can move onto the analysis protocol for the alignment and detection of RNA modifications using m6ANet or Dorado. At the end of this protocol, I've also included a Troubleshooting section to cover some common issues I've had while sequencing to hopefully streamline your runs. Where possible, I have highlighted deviations from the original protocol in bold red terms to make it easy to identify changes.

Requirements

Below is a list of required reagents you will need to carry out the protocol.

Materials and Equipment

Type	Items
Materials	50 ng of poly(A)-tailed RNA or 500 ng of total RNA in 8 μL. Note: For my best run, it was 333 ng in 9 μL of pure mRNA. RT Adapter (RTA, Blue Cap). RNA CS (RCS, Yellow Cap). RNA Adapter (RMX, Green Cap). Wash Buffer (WSB, Orange Cap). Elution Buffer (ELB, Black Cap).
Consumables	NEBNext® Quick Ligation Reaction Buffer (NEB, B6058). T4 DNA Ligase 2M U/mL (NEB, M0202T/M). 0.2 mL thin-walled PCR tubes. Nuclease-free water (e.g. ThermoFisher, AM9937). Agencourt RNAClean XP beads (Beckman Coulter™, A63987). Freshly prepared 70% ethanol in nuclease-free water. 1.5 mL Eppendorf DNA LoBind tubes. SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, 18080044). 10 mM dNTP solution (e.g. NEB N0447). Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851).
Equipment	Magnetic separator, suitable for 1.5 mL Eppendorf tubes. Hula mixer (gentle rotator mixer). Termal cycler.
Optional Equipment	Qubit fluorometer (or equivalent for QC check).

RNA Library Preparation

WARNING

You need to test your mRNA purity before proceeding. This can be done by saving some of the total RNA and running it on a gel (more information on interpreting those results here) as well as checking the concentration of total and mRNA on a nanodrop. You should have a decent concentration (with mRNA 3-6% of the measured total RNA) with 260/230 and 260/280 ratios of 2.0 or better (mor information on interpreting those results here). A low 260/230 ratio indicates organics in your sample, which can damage the flow cell pores, whereas a low 260/280 ratio indicates proteins, which can clog them. You may want to dilute your input sample so that you're loading your ideal amount in 8 μL of sample, which will also help dilute and contaminants. Higher mRNA amounts than expected (greater than 3-6% of the total RNA) suggests contamination by total RNA or DNA.

Regarding samples that have been frozen: I highly recommend that you pipette your sample 40+ times using an appropriate pipette to draw the full volume of the solution. You should also remeasure the concentration of your sample on the nanodrop, rather than relying on the concentration you obtained prior to freezing. I have had sample library preparations that were sub-par because the concentration dropped following a freeze/thaw.