NanoString Technologies, Inc. 1 PCR setup 2 Quick Start Guide MAN-10133-01 GeoMx—NGS RNA Library Prep Readout 1. Once Data Collection is complete, (see the GeoMx DSP Instrument Quick Start Guide), follow the prompts to finalize and unload the collection plate from the instrument. 2. Use a dry-down seal and your laboratory’s procedure to dry aspirates (i.e. dry at 65° C in thermal cycler with open top). 3. Rehydrate in 10 µL DEPC water, pipette up and down 5x, and let stand at RT for 10 minutes. Quick-spin. 1. Prep: Program a thermocycler with a 100°C heated lid according to table. Clean workspace with 10% bleach/RNase Away, distilled H 2 O, and 70% EtOH. Thaw GeoMx Seq Code Primer Plates (refer to lab worksheet for correct lettered plates) on ice and keep all other PCR reagents on ice. Quick-spin. 2. Add reagents to PCR plate, ensuring that Primer Plate, DSP collection plate and PCR plate are all in correct orientation (well A1 at upper left). Aliquot 2 μL 5x PCR Master Mix, then 4μL primer, then 4 μL DSP aspirate to each well of the new PCR plate. Pipette up and down 10x to mix. 3. Heat-seal plate. Pulse centrifuge to 1000 x g. 4. Transfer plate to thermocycler and run program. Unload plate 1 Step Temperature Time Cycles UDG incubation 37 O C 30 min 1X UDG deactivation 50 O C 10 min 1X Initial denaturation 95 O C 3 min 1X Denaturation 95 O C 15 sec Anneal 65 O C 60 sec 18X Extend 68 O C 30 sec Final extension 68 O C 5 min 1X Hold 4 O C ∞ 1X A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 DSP Plate Primer Plate (GeoMx Seq Code Pack Plate (A-H)) GeoMx Seq Code PCR Master Mix PCR Plate 2 μL 4 μL 4 μL
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NanoString Technologies, Inc. 1
PCR setup2
Quick Start Guide
MAN-10133-01GeoMx —NGS RNA Library Prep Readout
1. Once Data Collection is complete, (see the GeoMx DSP Instrument Quick Start Guide), follow the prompts to finalize and unload the collection plate from the instrument.
2. Use a dry-down seal and your laboratory’s procedure to dry aspirates (i.e. dry at 65° C in thermal cycler with open top).
3. Rehydrate in 10 µL DEPC water, pipette up and down 5x, and let stand at RT for 10 minutes. Quick-spin.
1. Prep: Program a thermocycler with a 100°C heated lid according to table. Clean workspace with 10% bleach/RNase Away, distilled H2O, and 70% EtOH. Thaw GeoMx Seq Code Primer Plates (refer to lab worksheet for correct lettered plates) on ice and keep all other PCR reagents on ice. Quick-spin.
2. Add reagents to PCR plate, ensuring that Primer Plate, DSP collection plate and PCR plate are all in correct orientation (well A1 at upper left). Aliquot 2 μL 5x PCR Master Mix, then 4μL primer, then 4 μL DSP aspirate to each well of the new PCR plate. Pipette up and down 10x to mix.
3. Heat-seal plate. Pulse centrifuge to 1000 x g.
4. Transfer plate to thermocycler and run program.
1. Transfer FASTQ files generated from sequencing to a location accessible by DND software.
2. Run the DND software (see the GeoMx—NGS DND User Manual.) This program will convert FASTQ files to DCCs.
3. Transfer the zipped DCC folder to a USB drive and insert it into the GeoMx DSP
4. In the GeoMx DSP Control Center, hover over the Data Collection button and select Upload Counts. Choose the appropriate zipped file.
Run DND and transfer data back to the GeoMx DSP system
1. Prepare a 1:8 dilution in a new tube by combining 2 ul of library and 16 ul of Elution Buffer.
2. Assess library quality of stock and 1:8 dilution quality using a capillary electrophoresis device such as the Agilent Bioanalyzer. Follow manufacturer instructions for use.
3. Check for the expected size of the amplicons and absence of primers, primer-dimers, or high molecular weight overamplification products. Expected library amplicon size is 162bp. Via Bioanalyzer, the library will appear as ~150bp.
Quality control4
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1. Libraries should be sequenced on Illumina® sequencing platforms with the following workflow specifications:
• Generate FASTQ • Dual-indexing • Paired-end with reads 2 x 27 • 5% PhiX spike-in by volume
2. Suggested loading concentrations are listed in the table above.
3. Set up the run according to the respective Illumina platform instructions (see respective Illumina platform user manuals at support.illumina.com).
Sequencing run setup5
Illumina platform Flow Cell Loading concentration
MiSeq V3 9 pM
NextSeq550 High-output 1.6 pM
NextSeq2000 P2 650 pM
Upload Counts
New / Continue Run
Data Collection
Target amplicon (150 bp)
Ideal library: Bioanalyzer DNA High Sensitivity trace
Additional Customer ResourcesFor more comprehensive information, visit us at nanostring.com and go to Support > Product Support to view manuals and other technical product literature.
NanoString Technologies, Inc. 530 Fairview Avenue NorthSeattle, Washington 98109