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 RNA Preparation
| Name | Recommended Manufacturer | Catalog # | Purpose |
|---|---|---|---|
| RNAeasy Plus Micro/Mini Kit | QIAGEN | 74034/74134 | RNA isolation from PBMC, sorted cells or tissues |
| Tempus Blood RNA tubes/Spin RNA isolation kit | Thermo Fisher | 4342792/4380204 | RNA isolation from whole blood |
| RNA 6000 Pico Kit for 2100 Bioanalyzer | Agilent | 5067-1511 | RNA quality control calculation |
| HS RNA Analysis Kit for Fragment Analyzer | Agilent | DNF-472_0502 | RNA quality control calculation |
Additional Reagents for Hybridization, cDNA Synthesis, Amplification, and Amplified Libraries QC
| Name | Recommended Manufacturer | Catalog # | Purpose |
|---|---|---|---|
| N-Lauroyl Sarcosine, sodium salt, 20% | Sigma | L7414 | Detergents for hybridization step in DirectCell protocol |
| 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 |
| Capillary Instrument or Slab Gel Electrophoresis System to analyze amplified DNA products |
||
| 2100 Bioanalyzer® | Agilent | |
| Fragment Analyzer™ | Agilent | |
| Agarose gel electrophoresis/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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