TITLE: GeoMx-NGS Readout Library Preparation 
AUTHORS: Angela R.S.  Kruse, Morad C Malek, Jamie Allen, Melissa Farrow, Jeff Spraggins

[DESCRIPTION]
This protocols describes the preparation of a library for Illumina NGS sequencing from Nanostring GeoMx DSP aspirate samples. 

[STEPS]
SECTION: Transferring the DSP Collection Plate
1. PREPARING THE COLLECTION PLATE (1 HOUR)
SECTION: Transferring the DSP Collection Plate
2. Remove the collection plate from the DSP instrument by following the instructions at the end of the GeoMx DSP run or clicking the plate status icon.
SECTION: Transferring the DSP Collection Plate
3. Seal the collection plate with a permeable membrane. if delaying library preparation for over 24 hours, seal with an nonpermeable membrane and store at -20°C. If shipping to another facility, seal with a nonpermeable membrane and wrap plate in bubble wrap and ship on dry ice (direct contact with dry ice may cause the plate to crack).
SECTION: Transferring the DSP Collection Plate
4. Dry down the collection plate by incubating on a thermocycler at 65°C for up to 1 hour. If there is still liquid in some of the wells after this time, dry down until all liquid has evaporated.
SECTION: Transferring the DSP Collection Plate
5. Spin down the plate and check that there is no liquid remaining prior to rehydrating the samples in the next step.
SECTION: Transferring the DSP Collection Plate
6. Rehydrate the samples. Add 10 μL of nuclease-free water, pipette up and down 5 times and allow the collected targets to solubilize for 10 minutes at room temperature. Use an adhesive plate seal to keep the sample from re-evaporating.
SECTION: Transferring the DSP Collection Plate
7. Pulse centrifuge the plate to 1000 x g to ensure all liquid has been collected at the bottom.
SECTION: Library Preparation
8. PCR setup (2 hours)

BEFORE YOU START

Program a thermocycler with a 100°C heated lid according to table (see Table 4).
Clean workspace with 10% bleach or RNase Away. Rinse with distilled H2O, followed by
70% EtOH.
Refer to the Lab Worksheet (see Figure 4) to choose the correct letter of GeoMx Seq Code Primer Plate for your DSP collection plate. Thaw the primer plate on ice or on cooling block.
  
Keep all PCR reagents as well as the PCR plate on ice or on cooling block during setup.
Once thawed, pulse centrifuge 5x PCR Master Mix, primer plate, and DSP collection plate to 1000 X g. Do not vortex at any point.

SECTION: Library Preparation
9. This PCR protocol begins with uracil-DNA glycosylase (UDG) incubation and deactivation.

 
            Step  Temp.  Time  Cycles    UDG   incubation  37 °C  30 min  1 X    UDG   deactivation  50 °C  10 min  1 X    Initial   denaturation  95 °C  3 min  1 X    Denaturation   Anneal    Extend  95 °C    65 °C    68 °C  15 sec    60 sec    30 sec  18 X    Final   extension  68 °C  5 min  1 X    Hold  4 °C  ∞  1 X  
 
SECTION: Library Preparation
10. PROCEDURE

The instructions here assume a 96-well plate. If running more or fewer aspirates, adjust the procedure
and volumes accordingly.


SECTION: Library Preparation
11. Prepare a new 96- well PCR plate Table 5: PCR reaction according to the table (see Table 5) and the steps below. Use of a multichannel pipette is recommended. Ensure that each component is added to the bottom of the well and that your Primer Plate , DSP collection plate and PCR plate are all in the correct orientation (well A1 at upper left). 
 
        PCR   amplification reaction components  Volume (µL)    5x PCR Master Mix  2   µL    Primer   mix (F & R, 2.5 µM each)  4 µL    DSP   aspirate  4 µL    TOTAL   VOLUME per reaction  10 µL  
 
SECTION: Library Preparation
12. Add 2 μL of 5x PCR Master Mix to each well of the new PCR plate. It is acceptable to reuse pipette tips for each column during this step.
SECTION: Library Preparation
13. Transfer 4 μL of primer from each row/column of the GeoMx SeqCode primer plate to each row/column of the PCR plate. Use of a multichannel pipette is encouraged. Change pipette tips for every row/column.
SECTION: Library Preparation
14. Add 4 μL of DSP aspirate from each row/column of the DSP collection plate to each row/column of the PCR plate. Change pipette tips for every row/column.
SECTION: Library Preparation
15. Pipette up and down 10 times to mix.
SECTION: Library Preparation
16. Heat-seal PCR plate according to manufacturer instructions. Pulse centrifuge to 1000 x g.
SECTION: Library Preparation
17. Incubate PCR plate in a thermocycler with program specified in Table 1(see Table 4). 

The PCR plate may be stored at 4°C overnight, or -20°C for up to 72 hours.
SECTION: Pooling and AMPure cleanup (45 minutes)
18. IMPORTANT: The following are post-PCR steps and should be performed in a separate lab space
from the pre-PCR steps.

Pool and purify PCR products using the steps below. The instructions below are for a single 96-well plate.


SECTION: Pooling and AMPure cleanup (45 minutes)
19. Once PCR is complete, pulse centrifuge PCR plate to 1000 x g.
SECTION: Pooling and AMPure cleanup (45 minutes)
20. Pool 4 μL of each PCR well into one 1.5 mL tube (see Figure 6). 

Using a multichannel pipette (8-channel), combine each column of samples into one 8-well strip tube. Once all columns have been combined into the strip tube, the contents of each strip tube well can then be pooled into a single 1.5 mL tube. If running multiple 96-well plates, pool each plate into its own 1.5 mL tube. However, if less than 12 reactions are on a plate, you may combine that pool with another plate's pool.
SECTION: Pooling and AMPure cleanup (45 minutes)
21. Measure the volume in the 1.5 mL tube using a pipette.
SECTION: Pooling and AMPure cleanup (45 minutes)
22. Add 1.2x volume of the measured volume of AMPure XP beads to the pooled PCR product. Mix well by pipetting up and down 10x. Pulse centrifuge. 

It is important to measure the exact volume of the pool. 

Example: pooling 4 μL from 96 wells of PCR product results in a ~384 μL pool. Measure the actual volume using a pipette, then add 1.2x this measured volume of AMPure XP beads to the pool.
SECTION: Pooling and AMPure cleanup (45 minutes)
23. Incubate 5 minutes at room temperature.
SECTION: Pooling and AMPure cleanup (45 minutes)
24. Prepare 20 mL fresh 80% EtOH by combining 16 mL 100% EtOH and 4 mL PCR-grade H2O.
SECTION: Pooling and AMPure cleanup (45 minutes)
25. Pellet beads on a magnetic stand for 5 minutes or until the solution is clear.
SECTION: Pooling and AMPure cleanup (45 minutes)
26. Carefully remove the supernatant. Avoid disturbing the beads, as they contain the library at this stage.
SECTION: Pooling and AMPure cleanup (45 minutes)
27. Wash beads with 1 mL of freshly-prepared 80% ethanol, incubating on magnetic stand for 30 seconds.
SECTION: Pooling and AMPure cleanup (45 minutes)
28. Discard the supernatant, being careful not to disturb the beads.
SECTION: Pooling and AMPure cleanup (45 minutes)
29. Wash beads a second time with 1 mL of freshly- prepared 80% ethanol, incubating on magnetic stand for 30 seconds.
SECTION: Pooling and AMPure cleanup (45 minutes)
30. Discard the supernatant, being careful not to disturb the beads.
SECTION: Pooling and AMPure cleanup (45 minutes)
31. Visually inspect the beads to ensure that as much ethanol as possible is removed without disturbing the beads. Use a low volume pipette (e.g., P20) to remove any residual ethanol, if necessary.
SECTION: Pooling and AMPure cleanup (45 minutes)
32. Dry beads on magnetic stand for ≤ 5 minutes. Remove from the magnetic stand.
SECTION: Pooling and AMPure cleanup (45 minutes)
33. Resuspend in 54 μL Elution Buffer. Mix well by pipetting.
SECTION: Pooling and AMPure cleanup (45 minutes)
34. Pellet beads on a magnetic stand for 5 minutes until the solution is clear.
SECTION: Pooling and AMPure cleanup (45 minutes)
35. Extract 50 μL of supernatant to a new tube. Leave 2–4 μL of supernatant in tube, if necessary, to avoid disturbing beads. This supernatant contains the sequencing library.
SECTION: Pooling and AMPure cleanup (45 minutes)
36. Add 60 μL of Ampure XP beads to the 50 μL supernatant. Mix well by pipetting up and down 10x. Pulse centrifuge.
SECTION: Pooling and AMPure cleanup (45 minutes)
37. Incubate 5 minutes at room temperature.
SECTION: Pooling and AMPure cleanup (45 minutes)
38. Pellet beads on a magnetic stand 5 minutes or until the solution is clear.
SECTION: Pooling and AMPure cleanup (45 minutes)
39. Carefully remove the supernatant. Avoid disturbing the beads.
SECTION: Pooling and AMPure cleanup (45 minutes)
40. Wash beads with 1 mL of freshly-prepared 80% ethanol, incubating on magnetic stand for 30 seconds.
SECTION: Pooling and AMPure cleanup (45 minutes)
41. Discard the supernatant, being careful not to disturb the beads.
SECTION: Pooling and AMPure cleanup (45 minutes)
42. Wash beads a second time with 1 mL of freshly- prepared 80% ethanol, incubating on magnetic stand for 30 seconds.
SECTION: Pooling and AMPure cleanup (45 minutes)
43. Discard the supernatant, being careful not to disturb the beads. 
SECTION: Pooling and AMPure cleanup (45 minutes)
44. Visually inspect to ensure that as much ethanol as possible is removed without disturbing the beads. Use a low volume pipette (e.g., P20) to remove any residual ethanol, if necessary.
SECTION: Pooling and AMPure cleanup (45 minutes)
45. Dry beads on magnetic stand for ≤ 5 minutes. Remove from magnetic stand.
SECTION: Pooling and AMPure cleanup (45 minutes)
46. Resuspend beads in Elution Buffer according to the table below . Mix well by pipetting.

 
 
        #   reactions  Elution Buffer   volume (µL)    96  48   µL    48  36 µL    24  24 µL    12  18 µL  
 


SECTION: Pooling and AMPure cleanup (45 minutes)
47. Incubate 5 minutes at room temperature.
SECTION: Pooling and AMPure cleanup (45 minutes)
48. Pellet the beads on the magnetic stand 5 minutes or until the solution is clear.
SECTION: Pooling and AMPure cleanup (45 minutes)
49. Extract the supernatant to a new tube. Leave 2 μL of supernatant in tube, if necessary, to avoid disturbing beads. This supernatant contains the sequencing library.

STOPPING POINT: The purified library may be stored in a DNA LoBind tube at -20°C until ready for sequencing.
SECTION: Quality control
50. Combine 2 μL of library and 14 μL of Elution Buffer to create a 1:8 dilution .
SECTION: Quality control
51. Assess quality of library stock and 1:8 dilution quality using a capillary electrophoresis device such as the Agilent Bioanalyzer. Follow manufacturer instructions for use.
SECTION: Quality control
52. Check for the expected size of the amplicons and absence of primers, primer-dimers, or high molecular weight overamplification products (see Figure 8). 

Expected library amplicon size is 162 bp. Using the Agilent Bioanalyzer, the library will appear as ~150bp. 

Verify that the concentration of the ~150bp amplicon peak of either the undiluted library or the 1:8 dilution falls within the Bioanalyer assay’s quantitative range (5 – 500 pg/ul). Use the molarity value (pM) from the Bioanalyzer to calculate library concentration.

  

SECTION: Sequencing Setup
53. 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

Use the Sample Index List downloaded from the GeoMx DSP Instrument. Copy and paste the Sample Index 
List into your Illumina Sample Sheet. Confirm the sequencing details entered here. If corrections 
are needed, you may correct the sheet and re-download the readout package, which includes the 
Sample Index List.

Set up the run according to the respective Illumina platform instructions (see respective Illumina 
platform user manuals at support.illumina.com). Suggested loading concentrations are listed below
(see Table 7).

  
          Illumina   platform  Flow cell  Loading   concentration    MiSeq  v3  9   pM    NextSeq550  High-output  1.6 pM    NextSeq2000  P2  650 pM  
 These values may be adjusted empirically based on internal workflow/specific sites/instruments.

SECTION: Transferring from the Illumina NGS Run
54. Transferring the NGS raw data (FASTQ) files

Sequencing output FASTQ files will need to be transferred and processed by NanoString's GeoMx
NGS Pipeline (DND) software program before being uploaded back to the GeoMx DSP system for
Data Analysis. See the GeoMx-NGS DND Software User Manual for additional guidance.