Depending on the type of samples and procedural options followed in the protocol, several of the following items will be required to run the assay.
For General DNA Preparation
Name | Recommended Manufacturer | Catalog # | Purpose |
---|---|---|---|
DNeasy Blood & Tissue Kit | QIAGEN | 69506 | DNA isolation from PBMC, T/B cells or tissues |
Tempus Blood RNA tubes/Spin RNA isolation kit | Thermo Fisher | 4342792/4380204 | DNA and RNA isolation from whole blood |
PAXgene Blood DNA kit/tubes | Qiagen | 761133/761165 | DNA isolation from whole blood |
All Prep DNA/RNA FFPE kit | Qiagen | 80234 | DNA and RNA isolation from FFPE tissues |
Additional Reagents for cDNA Synthesis, Amplification and Amplified Libraries QC
Name | Recommended Manufacturer | Catalog # | Purpose |
---|---|---|---|
Agencourt® AMPure® XP (Magnetic Beads) | Beckman-Coulter | A63881 | RNA-RevGSP hybrid and PCR product purification |
Dynabeads® MPC®-S (Magnetic Particle Concentrator) for 1.5 ml Test Tubes Magnetic Stand-96 for 96-well PCR plates |
Thermo Fisher | A13346/AM10027 | RNA-RevGSP hybrid and PCR product purification |
96-well PCR plates | Thermo-Fisher | A43673 | RT-PCR reactions |
Clear Adhesive Film for 96-well plates | Bio-Rad | MSB1001 | PCR plate seal |
High Sensitivity DNA Kit for Bioanalyzer | Agilent | 5067-4626 | PCR product QC/ quantification |
High Sensitivity NGS Fragment Analysis Kit (1 bp – 6000 bp) for Fragment Analyzer | Agilent | DNF-474-1000 | PCR product QC/ quantification |
Qubit® dsDNA HS (High Sensitivity) Assay Kit | Thermo Fisher | Q32851 | Quantify Indexed Libraries for NGS |
NextSeq® 500/550 High Output Kit v2.5 (300 cycles) /NovaSeq | Illumina | 20024908 (NextSeq) |
Next-Gen Sequencing |
Recommended Instrumentation
This protocol is optimized for the instruments specified below. Some modifications and optimization to the protocol may be necessary if using different models or instruments from other manufacturers.
Description | Model | Manufacturer |
---|---|---|
Spectrophotometer (NanoDrop or equivalent) | NanoDrop® ND-2000 | Thermo Fisher |
Thermal Cycler (Veriti 96-Well or equivalent) | Veriti® 96-Well | Thermo Fisher |
Instrument or Gel System to Analyze DNA Fragmentation |
2100 Bioanalyzer® | Agilent |
Fragment Analyzer™ | Agilent | |
Standard gel imaging system | many suppliers | |
DNA Quantification | Qubit 3.0 Fluorometer | Thermo Fisher |
Next-Generation Sequencer | NextSeq, NovaSeq | Illumina |
Thermomixer R (for microsampling AIR profiling) | Thermomixer R with heat block for 1.5-ml test tubes | Eppendorf/Fisher Scientific |
Other than the specific reagents and instruments listed above, the protocol assumes the user has access to standard materials (e.g., polypropylene tubes, pipette tips), equipment (tabletop centrifuges, pipettes), and common reagents (e.g., TE buffer, ethanol) and buffers used in a typical life science laboratory.
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