quick start guide geomx—ngs rna library prep...

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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.

Unload plate1

Step Temperature Time Cycles

UDG incubation 37O C 30 min 1X

UDG deactivation 50O C 10 min 1X

Initial denaturation 95O C 3 min 1X

Denaturation 95O C 15 sec

Anneal 65O C 60 sec 18X

Extend 68O C 30 sec

Final extension 68O C 5 min 1X

Hold 4O C ∞ 1X

ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12

DSP PlatePrimer Plate (GeoMx Seq Code Pack Plate (A-H))

GeoMx Seq CodePCR Master Mix

PCR Plate

2 μL 4 μL 4 μL


Quick Start GuideGeoMx —NGS RNA Library Prep Readout MAN-10133-01

The following assumes 96 well plate; adjust as needed

1. Pulse centrifuge PCR plate to 1000 x g. Combine 4 uL of each PCR product well into a single microtube.

2. Follow the steps in the diagram to purify pooled PCR products.

Pooling and AMPure cleanup 3

Transfer supernatantto 1.5 mL tube.

Dry on magnetic stand ≤5 min.

I ncubate beads on magnetic stand 30 sec.

Discard supernatant.

Pellet beads on magnetic stand 5 min.

Discard supernatant (beads contain the library).

Add AMPure beads (1.2x measured volume of

pooled PCR products). Pipette to mix, pulse

centrifuge. Incubate 5 min.

Pool 4 μL each PCR well into 1.5 mL tube. Measure

final volume.

Pellet beads on magnetic stand 5 min. The library is now in the supernatant.

Resuspend beads in 48 μL Elution Bu�er (for 96 well

plate). Incubate 5 min.

Dry on magnetic stand ≤5 min.

RepeatOnce

RepeatOnce

Incubate beads on magnetic stand 30 sec.

Discard supernatant.

Wash beads on magnetic stand with 200 µL 80% ethanol

Wash beads on magnetic stand with 200 µL 80% ethanol

Pellet beads on magnetic stand 5 min. Discard

supernatant

Add 60 μL AMPure beads Pipette to mix,

pulse centrifuge. Incubate 5 min.

Remove from magnetic stand. Resuspend in 50 μL Elution Bu�er.

Pipette to mix.



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

6

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

No residual primer detected

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. © 2020 NanoString Technologies, Inc. All rights reserved. NanoString, NanoString Technologies, the NanoString logo, nCounter, and GeoMx are registered trademarks or trademarks of NanoString Technologies, Inc. in the United States and/or other countries.

JUN 2020 MK2550-10

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

T (888) 358-6266F (206) 378-6288

nanostring.com [email protected]

For more information, please visit nanostring.com


quick start guide geomx—ngs rna library prep...

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