Direct RNA sequencing - sequence-specific (SQK-RNA004) (DSS_9197_v4_revD_02Apr2025)

DateVersionLanguageDeviceChanges
4/2/2025DSS_9197_v4_revD_02Apr2025EnglishGridION
2nd April 2025 - Early access was removed from protocol. - In 'Steps in the sequencing workflow', under 'Library preparation', timings in the Figure were edited to align with the Table. - In 'Direct RNA Sequencing Kit (SQK-RNA004) contents', the RNA CS (RCS) described previously as the 'calibration' strand was changed to 'control' strand in the note under the Table. - Consumables and equipment available from Oxford Nanopore were included in Section 2, 'Equipment and consumables'. - Hyperlink for DNA/RNA handling was updated in 'Input RNA'. - Addition of a Checkpoint to 'Perform a flow cell check' in Section 3, 'Library Preparation' and in Step 1, to ensure that the T4 DNA Ligase is kept on ice and not thawed. - Steps 9, 12, and 37 were edited for clarity. - For flow cell image 1a in 'Priming and loading the MinION/GridION Flow Cell', the word 'DNA' was removed from library. - For the PromethION branch, the Step for removing PromethION Flow Cell out of the fridge was moved to the beginning of the Section on 'Priming and loading the PromethION Flow Cell' to enable the flow cell to reach room temperature while the flow cell priming mix is prepared. And an optional action of performing a flow cell check was included. - In Section 5, 'Data acquisition and basecalling', 'To start a sequencing run in MinKNOW', the specific RNA kit was specified in point 3, 'Select the Direct RNA Sequencing Kit (SQK-RNA004) on the Kit selection page'. - The function of the Flow Cell Wash Kit in Section 6, 'Flow cell reuse and returns' was edited for clarity.
4/2/2025DSS_9197_v4_revD_02Apr2025EnglishMinION
2nd April 2025 - Early access was removed from protocol. - In 'Steps in the sequencing workflow', under 'Library preparation', timings in the Figure were edited to align with the Table. - In 'Direct RNA Sequencing Kit (SQK-RNA004) contents', the RNA CS (RCS) described previously as the 'calibration' strand was changed to 'control' strand in the note under the Table. - Consumables and equipment available from Oxford Nanopore were included in Section 2, 'Equipment and consumables'. - Hyperlink for DNA/RNA handling was updated in 'Input RNA'. - Addition of a Checkpoint to 'Perform a flow cell check' in Section 3, 'Library Preparation' and in Step 1, to ensure that the T4 DNA Ligase is kept on ice and not thawed. - Steps 9, 12, and 37 were edited for clarity. - For flow cell image 1a in 'Priming and loading the MinION/GridION Flow Cell', the word 'DNA' was removed from library. - For the PromethION branch, the Step for removing PromethION Flow Cell out of the fridge was moved to the beginning of the Section on 'Priming and loading the PromethION Flow Cell' to enable the flow cell to reach room temperature while the flow cell priming mix is prepared. And an optional action of performing a flow cell check was included. - In Section 5, 'Data acquisition and basecalling', 'To start a sequencing run in MinKNOW', the specific RNA kit was specified in point 3, 'Select the Direct RNA Sequencing Kit (SQK-RNA004) on the Kit selection page'. - The function of the Flow Cell Wash Kit in Section 6, 'Flow cell reuse and returns' was edited for clarity.
4/2/2025DSS_9197_v4_revD_02Apr2025EnglishPromethION
2nd April 2025 - Early access was removed from protocol. - In 'Steps in the sequencing workflow', under 'Library preparation', timings in the Figure were edited to align with the Table. - In 'Direct RNA Sequencing Kit (SQK-RNA004) contents', the RNA CS (RCS) described previously as the 'calibration' strand was changed to 'control' strand in the note under the Table. - Consumables and equipment available from Oxford Nanopore were included in Section 2, 'Equipment and consumables'. - Hyperlink for DNA/RNA handling was updated in 'Input RNA'. - Addition of a Checkpoint to 'Perform a flow cell check' in Section 3, 'Library Preparation' and in Step 1, to ensure that the T4 DNA Ligase is kept on ice and not thawed. - Steps 9, 12, and 37 were edited for clarity. - For flow cell image 1a in 'Priming and loading the MinION/GridION Flow Cell', the word 'DNA' was removed from library. - For the PromethION branch, the Step for removing PromethION Flow Cell out of the fridge was moved to the beginning of the Section on 'Priming and loading the PromethION Flow Cell' to enable the flow cell to reach room temperature while the flow cell priming mix is prepared. And an optional action of performing a flow cell check was included. - In Section 5, 'Data acquisition and basecalling', 'To start a sequencing run in MinKNOW', the specific RNA kit was specified in point 3, 'Select the Direct RNA Sequencing Kit (SQK-RNA004) on the Kit selection page'. - The function of the Flow Cell Wash Kit in Section 6, 'Flow cell reuse and returns' was edited for clarity.
12/11/2024DSS_9197_v4_revC_11Dec2024EnglishGridION
11th Dec 2024 - Updated kit content to include information on different RCS vial concentration in new kit batches. - Addition of links to MinION Mk1D IT requirements and user manual. - Arrow which was previously at the bottom edge of the flow cell in Image 1b "Priming and loading MinION/GridION Flow Cell" was moved to the middle of the flow cell. - Updated link for flow cell returns in "Flow cell reuse and returns" and removed note for flushing flow cells. - Updated link for EPI2ME in "Downstream analysis".
12/11/2024DSS_9197_v4_revC_11Dec2024EnglishMinION
11th Dec 2024 - Updated kit content to include information on different RCS vial concentration in new kit batches. - Addition of links to MinION Mk1D IT requirements and user manual. - Arrow which was previously at the bottom edge of the flow cell in Image 1b "Priming and loading MinION/GridION Flow Cell" was moved to the middle of the flow cell. - Updated link for flow cell returns in "Flow cell reuse and returns" and removed note for flushing flow cells. - Updated link for EPI2ME in "Downstream analysis".
12/11/2024DSS_9197_v4_revC_11Dec2024EnglishPromethION
11th Dec 2024 - Updated kit content to include information on different RCS vial concentration in new kit batches. - Addition of links to MinION Mk1D IT requirements and user manual. - Arrow which was previously at the bottom edge of the flow cell in Image 1b "Priming and loading MinION/GridION Flow Cell" was moved to the middle of the flow cell. - Updated link for flow cell returns in "Flow cell reuse and returns" and removed note for flushing flow cells. - Updated link for EPI2ME in "Downstream analysis".
7/31/2024DSS_9197_v4_revB_20Sep2023EnglishGridION
31 July 2024 - Updated the protocol format to the new "slimmed-down" introduction sections. - Updated workflow image in introduction to fix formatting errors in PDF generation - Updated the Reverse transcriptase reagents and steps to the new method using Induro® Reverse Transcriptase (NEB, cat # M0681). - Updated the RNase Inhibitor reagents and steps to the new method using Murine RNase Inhibitor (NEB, M0314). - Updated first Agencourt RNAClean XP bead clean in Library Preparation section to elute into 23 ul Nuclease-free water, retain the full 23 ul and no longer have to add 3 ul of water in the next step. - Updated "Flow cell reuse and returns" section to specify compatibility and use-cases for the Flow Cell Wash Kit with the RNA sequencing kit and RNA flow cells.
7/31/2024DSS_9197_v4_revB_20Sep2023EnglishMinION
31 July 2024 - Updated the protocol format to the new "slimmed-down" introduction sections. - Updated workflow image in introduction to fix formatting errors in PDF generation - Updated the Reverse transcriptase reagents and steps to the new method using Induro® Reverse Transcriptase (NEB, cat # M0681). - Updated the RNase Inhibitor reagents and steps to the new method using Murine RNase Inhibitor (NEB, M0314). - Updated first Agencourt RNAClean XP bead clean in Library Preparation section to elute into 23 ul Nuclease-free water, retain the full 23 ul and no longer have to add 3 ul of water in the next step. - Updated "Flow cell reuse and returns" section to specify compatibility and use-cases for the Flow Cell Wash Kit with the RNA sequencing kit and RNA flow cells.
7/31/2024DSS_9197_v4_revB_20Sep2023EnglishPromethION
31 July 2024 - Updated the protocol format to the new "slimmed-down" introduction sections. - Updated workflow image in introduction to fix formatting errors in PDF generation - Updated the Reverse transcriptase reagents and steps to the new method using Induro® Reverse Transcriptase (NEB, cat # M0681). - Updated the RNase Inhibitor reagents and steps to the new method using Murine RNase Inhibitor (NEB, M0314). - Updated first Agencourt RNAClean XP bead clean in Library Preparation section to elute into 23 ul Nuclease-free water, retain the full 23 ul and no longer have to add 3 ul of water in the next step. - Updated "Flow cell reuse and returns" section to specify compatibility and use-cases for the Flow Cell Wash Kit with the RNA sequencing kit and RNA flow cells.
July 31st, 2024DSS_9197_v4_revA_20Sep2023EnglishPromethion Download -
July 31st, 2024DSS_9197_v4_revA_20Sep2023EnglishGridion Download -
July 31st, 2024DSS_9197_v4_revA_20Sep2023EnglishMinion Download -