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.
Additional Reagents and Apparatus for Hybridization, cDNA Synthesis, Amplification, and Amplified Libraries QC
| Name | Recommended Manufacturer | Catalog # | Purpose |
|---|---|---|---|
| SPRIselect, 5 ml (Magnetic Beads) | Beckman Coulter | B23317 | RNA-RevGSP hybrid and PCR product purification |
| Dynabeads® MPC®-S (Magnetic Particle Concentrator) for 1.5 ml Test Tubes | Thermo Fisher | A13346 | RNA-RevGSP hybrid product purification |
| Catalyst 96 slotted ring magnetic plate | ALPAQUA | A000550 | RNA-RevGSP hybrid product purification in 96-well plate |
| 96-well PCR plates | Thermo Fisher | A43673 | RT-PCR reactions |
| Clear Adhesive Film for 96-well plates | Bio-Rad | MSB1001 | PCR plate seal |
| 8-channel pipettor | various | various | To pipette 8-rows across when setting up reactions |
| 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® 1000/2000 XLeap-SBS P1 Reagent Kit (600 cycles) | Illumina | 20100981 | Next-Gen Sequencing |
| NextSeq® 1000/2000 XLeap-SBS Read Primer Kit | Illumina | 20112859 | Next-Gen Sequencing with custom primers |
| MiSeq Reagent Kit v3 (600 cycles) | Illumina | MS-102-3003 | Next-Gen Sequencing |
| MiSeq i100 Series 25M Reagent Kit (600 cycles) | Illumina | 20115696 | 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 Agarose Gel System to analyze DNA fragments 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 2000, MiSeq | Illumina |
| Thermomixer R | Thermomixer R or MixPlate with lock for vortexing PCR plates | Eppendorf/Fisher Scientific |
| FisherBrand Mini Plate Spinner Centrifuge | Centrifugation of plate to collect droplets from walls | 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.
Need more help with this?
Contact Us