Modified nuclease flush protocol for Nanopore RNA flow cells v1

Résumé fourni par la source

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. 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. 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.

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