Jul 03, 2024

Public workspaceModified nuclease flush protocol for Nanopore RNA flow cells 

DOI
dx.doi.org/10.17504/protocols.io.6qpvr8p6plmk/v1
  • 1University of Melbourne
Anthea Hull
Open access
DOI: dx.doi.org/10.17504/protocols.io.6qpvr8p6plmk/v1
Protocol Citation: Anthea Hull, Ricardo De Paoli-Iseppi, Mike Clark 2024. Modified nuclease flush protocol for Nanopore RNA flow cells . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8p6plmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 01, 2024
Last Modified: July 03, 2024
Protocol Integer ID: 102634
Keywords: Nanopore, RNase flush, RNA, Flow cell, long reads
Disclaimer
- This method was developed on PromethION FLO-PRO004RA flow cells and has not been tested in other systems.
- All results shown are for runs sequencing total RNA. PolyA+ runs, or runs experiencing less pore blocking, may see smaller improvements after flowcell flushing.
Abstract
A nuclease flush can be used to recover blocked pores on Oxford Nanopore Technologies (ONT) flow cells to maximise overall sequencing output. The Flow Cell Wash Kit (EXP-WSH004/EXP-WSH004-XL) was developed to remove blockages on DNA flow cells, but does not restore blocked pores on their RNA counterparts. This short protocol describes a simple modification to the Nanopore Flow Cell Flush protocol to enable pore recovery in RNA flow cells.


Figure 1. Summary of mean number of available and unavailable pores during RNA sequencing before and after RNase flush (n=9). Progressive blocking leads to increasing unavailable pores, which can be partially returned to their original available state following nuclease flush. Error bars are standard deviations.
Figure 1. Summary of mean number of available and unavailable pores during RNA sequencing before and after RNase flush (n=9). Progressive blocking leads to increasing unavailable pores, which can be partially returned to their original available state following nuclease flush. Error bars are standard deviations.

This protocol uses two RNases to digest blockage-causing nucleic acids: RNase CocktailTM Enzyme Mix and RNase H. RNase CocktailTM contains endoribonucleases RNase A and RNase T1, which cleave at C and U, and G residues, respectively. RNase H, rather than cleaving single-stranded RNA, degrades the RNA strand of DNA-RNA hybrid molecules, allowing it to target the hybrid libraries used for direct RNA sequencing. When used in combination with WMX (a DNase I containing solution provided in the ONT EXP-WSH004 wash kit), blockage-causing DNA, single-stranded RNA or DNA-RNA hybrids can be degraded.

Figure 2. Effect of WMX in RNase Flow Cell Wash Mix on recovery of available pores. Flushing with WMX and RNase (RNase CocktailTM + RNase H) together (n = 9) increases the % of available pores beyond the effects of flushing with RNase alone (n = 3). Data presented as mean and standard deviations.
Figure 2. Effect of WMX in RNase Flow Cell Wash Mix on recovery of available pores. Flushing with WMX and RNase (RNase CocktailTM + RNase H) together (n = 9) increases the % of available pores beyond the effects of flushing with RNase alone (n = 3). Data presented as mean and standard deviations.

Based on our preliminary results, this protocol can recover up to 50% of available pores present at the start of a run, and can boost the number of sequenced bases by >50%. Additionally, flushing RNA flow cells in accordance with this protocol has a negligible effect on read quality or length.
Figure 3. Summary of pore availability immediately pre- and proceeding RNase flush (with and without the addition of WMX), and resulting sequencing yields from pre- and post-flush runs. Presented as mean (standard deviation). Flushing with RNase and WMX together leads to substantial available pore recovery, and provides a corresponding boost in generated sequencing data with a minimal drop in read quality or n50. Note: Data was generated from 12 FLO-PRO004RA flow cells sequencing total RNA, each flushed once, and reloaded with a new total RNA library. Libraries sequenced before and after flush were generated on different days from the same RNA sample, and different RNA samples were used for each flow cell.
Figure 3. Summary of pore availability immediately pre- and proceeding RNase flush (with and without the addition of WMX), and resulting sequencing yields from pre- and post-flush runs. Presented as mean (standard deviation). Flushing with RNase and WMX together leads to substantial available pore recovery, and provides a corresponding boost in generated sequencing data with a minimal drop in read quality or n50.

Note: Data was generated from 12 FLO-PRO004RA flow cells sequencing total RNA, each flushed once, and reloaded with a new total RNA library. Libraries sequenced before and after flush were generated on different days from the same RNA sample, and different RNA samples were used for each flow cell.

Materials
Flow Cell Wash Kit (EXP-WSH004) or Flow Cell Wash Kit XL (EXP-WSH004-XL) from Oxford Nanopore Technologies (ONT).
RNase H (NEB, M0297)
RNase CocktailTM Enzyme Mix (Invitrogen, AM2286)

Protocol
Protocol
Thaw Wash Mix (WMX), RNase H and RNase CocktailTM on ice. Do not vortex.
Thaw one tube of Wash Diluent (DIL) at room temperature.
Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, then spin down briefly and place on ice.
In a fresh 1.5 ml Eppendorf DNA LoBind tube, prepare the following Flow Cell Wash Mix:

ReagentVolume per flow cell
Wash Mix (WMX)2 μl
RNase H1 μl
RNase Cocktail™1 μl
Wash Diluent (DIL)396 μl
Total400 μl

Mix well by pipetting, and place on ice. Do not vortex the tube.
Follow Oxford Nanopore EXP-WSH004/EXP-WSH004-XL Protocol (accessible from link below) from Step 6 as directed, using the prepared Flow Cell Wash Mix to complete the flow cell flush.
EXP-WSH004/EXP-WSH004-XL Protocol:
https://community.nanoporetech.com/docs/prepare/library_prep_protocols/flow-cell-wash-kit-exp-wsh004/v/wfc_9120_v1_revq_08dec2020/flushing-a-promethion-flow-cell?devices=promethion
Optional: Following the 1 hour incubation and waste removal steps as described in the standard protocol, we recommend the flow cell be loaded with storage buffer as detailed in the "To store the Flow Cell for later use" protocol and a flow cell check be performed to quantify extent of pore recovery.

Flow Cell storage Protocol:
https://community.nanoporetech.com/docs/prepare/library_prep_protocols/flow-cell-wash-kit-exp-wsh004/v/wfc_9120_v1_revq_08dec2020/to-store-the-promethion-flow-cell-for-later-use?devices=promethion
Optional
The flow cell may now be stored for later use or used immediately with a fresh RNA library.
Protocol references