Nanodigmbio-NadPrep Total RNA-To-DNA Module-Protocol
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NadPrep Total RNA-To-DNA Module User Manual V1.1 Applicable to NadPrep Total RNA-To-DNA Module Thoroughly Read This Manual Before Operation. For Research Use Only. Version1.1

For research use only. Not for use in diagnostic procedures. This instruction is intended for use with the NadPrep Total RNA-To-DNA Module. Nanodigmbio Pte. Ltd. reserves all rights. Version Data Released Change 1.1 Nov 2023 Revise description of storage temperatures for kit and components. For Research Use Only. 2/14 Nanodigmbio Pte. Ltd.

Step 2: 1st Strand Synthesis (RNA (Level A)) 9 Step 1: Random Primer Hybridization (RNA (Level B)) 9 Step 2: 1st Strand Synthesis (RNA (Level B)) 10 Nanodigmbio Pte. Ltd. 3/14 For Research Use Only.

• Physically separate the laboratory space and establish an operating area for RNA handling, with disinfecting regularly. All the equipment and consumables are dedicated. • Gently pipet RNA rather than thoroughly vortex. • Place the RNA specimens on ice and operate immediately once outside the freezer to avoid degradation. • Make sure to proceed the experiment from RNA handling to 2nd strand synthesis by using RNase-free consumables and reagents. Gloves are essential around all steps. Replace new gloves once contacting the equipment or space out of the RNase-free area. • The personal...

NadPrep Total RNA-To-DNA Module is a total RNA reverse transcription module developed for high-throughput sequencing platform. This kit offers a stable and efficient library preparation solution for total RNA input ranging from 10 ng to 200 ng. This kit can be combined with NadPrep DNA Universal Library Preparation kit and downstream hybridization capture reagents from Nanodigmbio for detecting detailed biological information in targeted regions including gene expression, SNV, alternative splicing, fusion, etc. The RNA targeted sequencing procedure dispenses with rRNA elimination, ensuring...

Library C0nstructi0n NadPrep DNA Library Preparation Moudle (for DNBSEQTM), 24 rxn 1002211 NadPrep DNA Library Preparation Moudle (for DNBSEQTM), 96 rxn 1002212 Blocking Oligos Blocking Oligos (for DNBSEQTM, SI), 96 rxn 1006205 Blocking Oligos (for DNBSEQTM, DI), 96 rxn 1006208 Hybrid Capture Reagents NadPrep Hybrid Capture Reagents, 16 rxn 1005102 NadPrep Hybrid Capture Reagents, 96 rxn 1005101 Nad Panels Onco Plus Research Panel v2.0, 96 rxn 1001111E OnCT Research Panel v1.0, 96 rxn 1001841 For Research Use Only. 6/14 Nanodigmbio Pte. Ltd.

Adapter Blocking Post-amplification Cleanup Library Fragments Enrichment Hybridization Capture Targeted Capture Blocking Oligos NadPrep Hybrid Capture Reagents * Coupled with NadPrep DNA Library Preparation Module (for DNBSEQ™) as an example. Equipment and Consumable Equipment Item Digital electrophoresis Agilent 2100 Electrophoresis Bioanalyzer® system (Cat # G2939AA) Agilent 2200 Electrophoresis Bioanalyzer® system (Cat # G2965AA) Bioptic Qsep100 capillary gel electrophoresis system General laboratory supplier Benchtop centrifuge General laboratory supplier Thermal cycler Microcentrifuge...

RNA Input: 10-200 ng; Preferably quantified by Qubit™ fluorometer or a similar fluorometric method. RNA Purification: High quality RNA input, with OD260/OD280 = 1.8-2.0 and OD260/OD230 = 2-2.5. RNA Quality: The RNA sample requires additional QC before handling for the identification of integrality. The RNA can be classified into Level A (with intact RNA or partial intact RNA) and Level B (with highly degraded RNA) as follows: Cell or tissue FFPE tissue Cell or tissue FFPE tissue RNA RIN (RNA integrity number) is recommended for the assessment of integrality of RNA from cell or tissue. RNA...

1A. Thaw 1st Strand Synth. Enzyme on ice. Thoroughly vortex and briefly centrifuge to collect contents. 2A. Set up each reaction in a 0.2 mL PCR tube on ice as follows: Fragmented RNA & Random primers mix 10 ^L 1st Strand Synth. Enzyme 2 |iL Nuclease Free Water 8 |iL 3A. Mix thoroughly and then briefly centrifuge to collect contents. 4A. Incubate the tube in the thermal cycler programmed as described, with the heated lid set at 105°C. 1B. Thaw Random Primer on ice. Thoroughly vortex and briefly centrifuge to collect contents. 2B. Set up each reaction in a 0.2 mL PCR tube on ice as follows:...

1B. Thaw 1st Strand Synth. Buffer and 1st Strand Synth. Enzyme on ice. Thoroughly vortex and briefly centrifuge to collect contents. 2B. Set up each reaction in a 0.2 mL PCR tube on ice as follows: 1st Strand Synth. Buffer 4 |iL 1st Strand Synth. Enzyme 2 |iL Nuclease Free Water 8 |iL Total 20 ^L 3B. Mix thoroughly and then briefly centrifuge to collect contents. 4B. Incubate the tube in the thermal cycler programmed as described, with the heated lid set at 105°C. 1. Thaw 2nd Strand Synth. Buffer and 2nd Strand Synth. Enzyme on ice. Thoroughly vortex and briefly centrifuge to collect...

O Note: Equilibrate the NadPrep SP Beads to room temperature for a minimum of 30 min. Thoroughly vortex the beads for 45 seconds at high-speed. 1. Add 90 |iL of the NadPrep SP Beads to the same tube from Step 3. Mix thoroughly and then incubate at 25°Cfor 10 min . 2. Briefly centrifuge and then place the tube on the magnetic stand until the solution is clear (~5 min). Carefully remove and discard the supernatant without disturbing the beads. O Note: Adjust the incubating time according to the actual performance of different magnetic stands to ensure the solution completely clarified. 3....

The condition of fragmentation differs from RNA grades. This instruction defines the RNA quality into two grades: level A (with intact RNA or partial intact RNA) and level B (with highly degraded RNA). The fragmentation condition depends on the value of RIN and DV 200 of RNA samples (as shown in Fig 1 & 2). A Fig 1. Classification and fragmentation conditions of RNA from cell lines. A. Intact RNA from cell lines (RIN=7, Level A). The fragmentation can be performed at 94℃ for 15 min; B. Partial intact RNA from cell lines (RIN=4.9, Level A). The fragmentation can be performed at 94℃ for 8...