Lipid Nanoparticle Profiler Kit, PREP

This user guide is written to cover steps 1-4 when using ONI’s LNP Profiling workflow in an
automated format (also known as Lipid Nanoparticle Profiler Kit, PREP or LNP PREP) on the
Aplo Flow system:

Step 1: Define experiment and sample details
Step 2: Load reagents system deck
Step 3: Begin LNP Sample Preparation
Step 4: Automated Cargo Addition
Step 5: (Not covered in this guide) AutoLNP data acquisition and analysis

For more information regarding AutoLNP data acquisition and analysis, please refer to the ONI learning portal.

• The LNP PREP kit was optimized for a room temperature of 18˚C – 30˚C and humidity >25%. Contact ONI’s technical support if the working conditions do not meet the criteria.
• We recommend performing a dilution series across the RNA concentration range using a full Assay Chip (four lanes), running a full LNP Profiler Assay, and select the concentration that gives around 1000-2000 LNP counts/field of view on a Nanoimager or 3000-6000 LNP counts/field of view on an Aplo Scope.
• Final RNA concentration of diluted LNPs should be 40-100 ng/mL, assuming an encapsulation efficiency of over 50%.

LNP PREP utilizes a variable amount of pipette tips per run depending on the assay configuration. Please consult the tables below to estimate the number of pipette tips required, thus subsequent quantity of 384 tip pipette racks needed for your total LNP PREP.

The equipment and consumables listed below must be provided by the user.

Equipment

• Standard personal protective equipment such as gloves, safety glasses, and lab coat
• Biohazardous waste vessel as required by local health and safety regulations
• Pipettors (p2.5, p200, p1000)
• Benchtop microcentrifuge compatible with strips of 8 0.2 mL tubes at 5,000 RPM
• Humidity chamber (such as Simport Stain Tray M918-2TM)

Consumables

• LNP sample(s) to be tested
• Bead slide for channel mapping (ONI- 800-00016)
• Manual pipette tips (10 µL, 200 µL, 1000 µL)
• Lens cleaning paper
• Objective oil
• Lint-free cleaning wipes (Kimwipes)
• Alternative buffer for cargo staining (if desired)

1. If the wash buffer has been stored at -20°C, allow an additional hour before starting
   for it to thaw completely, or use a water bath to accelerate thawing.

   a. The wash buffer tube can be stored at 4°C to eliminate thaw time.

Before starting, verify that the Aplo Flow system is connected and connected to the software.

1. Open CSA and wait for status to change to “running”
2. Verify the System Status icon in the top right-hand corner of CODI shows a green checkmark.

1. 1. From the Chrome browser, navigate to alto.codi.bio
   2. Log into your CODI account
   3. Open the LNP PREP Experiment app from the “acquisition apps” tab on the left of the
      main CODI page ()

The center section of page #1 is used to define the sample configuration for each chip in the preparation.  Depending on the number of chips defined in the global assay settings, 1,2, 3, or 4 chips will be displayed at the top of the sample settings panel.

Click on the image of any chip to directly access its specific sample settings dialog- note the white outline indicating selection.  Alternatively, click on the arrows to the left or right of the chip diagram to toggle to the chip of choice and enter its sample settings.

For each chip, define the individual sample settings:

This page displays the required sample and reagent setup for the defined experiment and samples.  There is no data entry on this page, however, the layout dynamically updates based on how samples and chips are defined during step #1.

1. Use a benchtop centrifuge to spin down the Chip Strips, one per assay chip to be processed Do not remove the seal from the top of the Chip Strips.

2. Locate the side of each Chip Strip with a hole in the blue plastic frame (arrow in image below). Note that the orientation hole may not be visible from the top of the Chip Strip due to the seal placement. This indicates orientation and the hole MUST BE positioned on the right-hand side when facing the machine so that it mates with its  corresponding locating pin on the Reagent Block.

3. Load one Chip Strip for each chip to be processed in the workflow, corresponding to Chips A to D on the chip holder block. Ensure the Chip Strips are fully seated so they lie flat and flush with the surface of the Reagent Block.

5. (OPTIONAL) If the LNP reagent block was removed from the deck to load reagents, place it back on the Aplo Flow deck.  View the Reagent Block from the side to visually confirm it is fully seated flat against the deck.

Load the pipette tip box into position C of the Aplo Flow Deck with its lid on.  Similar to the reagent block, place the back edge of the tip box into the rear of the nest position, then press down on the lid to compress the retention springs and fully seat the tip box on the deck.

Remove Assay Chips (previously equilibrated to room temperature) from their shipping pouches and load each chip onto the chip holder block in Position E of the Aplo Flow Deck.

1. Empty the contents of the waste tub before each run in accordance with local health and safety guidance for pipette tips exposed to potential biological hazards.
2. Insert the empty waste tub in the recessed position on the deck, position F, with the lid on top. 

If “pause for manual sample addition” was checked on during step #1, skip this section for during initial setup, then dilute your LNPs once the system indicates it is paused. See Appendix section “Pause for LNP addition” for more details about this feature.

1. Dilute LNP samples in Wash Buffer or 1X PBS without calcium and magnesium. The exact dilution factor depends on the sample concentration.
2. Add at least the minimum volume of each sample to one of the provided PCR tubes.
   a. One tube of sample is required for each unique sample source defined in step #1. One tube can be used for multiple chips and lanes.
   b. Adding additional volume beyond the required minimum will not have an impact.
   c. For one chip, up to 4 separate samples can be loaded. For four chips, up to 16 separate samples can be loaded.
3. Place sample tube(s) in the position indicated on the reagent block platemap in step #2.

The table below outlines the general steps of the LNP PREP assay.  Depending on configuration, some steps are optional or may not be performed – for example ligand detection.  There are multiple incubation and delay steps not shown in the steps listed, at which time it is normal that the system may appear to be idle for an extended period.  This is expected behavior to properly time liquid addition steps and process multiple samples.

When the run has completed, a ‘Preparation Complete’ dialog will appear.  Note that depending on the experiment setup, not every tube of the Reagent Chip Strip will be pierced by the end of the run.

Except for a single chip ready for acquisition or automated cargo detection addition, remove all other chips from the holder block, seal with the provided stickers, and store at 4°C.  For prolonged storage (>2hrs) ONI recommends storing the sealed chips in a humidity chamber until imaged.

Once the protocol has finished running, carefully remove any Chip Strips and store them at 4°C until automated cargo detection addition has been performed.

• If not interrogating cargo in samples, any used Chip Strips may be disposed of at this point.
• If proceeding immediately to automated cargo detection addition for one chip, the Chip Strip which corresponds to the chip of interest may be left in position in the reagent block. Remove all the other Chip Strips and store them at 4°C until needed for automated cargo detection.

The LNP Profiler workflow includes the capability to interrogate cargo positivity of LNP samples.  Due to the time sensitive nature of cargo detection reagent once added to a chip, the reagent must be added to the sample directly before imaging, and imaging must occur within an hour of completing the cargo addition protocol.  Cargo detection reagent addition can be performed either manually (following the protocol found in the online user guide for Application Kit: LNP Profiler) or automated on a chip-by-chip basis by following the directions found in this section.

This page displays a fixed set of chips to individually add cargo detection reagent.  This page will show the number of chips defined during step #1.

1. Click the ‘Add cargo detection’ button next to one of the chips ready to be imaged.

Once the ‘Add cargo detection’ button has been clicked, one of two dialogs will appear depending on which buffer was selected:

1. The cargo detection reagent is already packaged in one of the unused tubes on the Chip Strip. Load ONLY the chip strip that corresponds to the chip selected for automated cargo detection reagent addition in the location indicated in the diagram.
2. Regardless of buffer selection, load the 15mL Nalgene bottle of LNP Wash Buffer in the position indicated in the diagram.
3. If processing cargo detection with User Defined Buffer, add at least 5mL of desired buffer to the empty 15mL Nalgene tube provided in the reagent kit and place the filled tube on the deck in the position indicated in the diagram.
4. Load ONLY the single corresponding chip to be processed for automation cargo detection addition in the position indicated in the diagram.  
5. Load a new rack of pipette tips to position C of the Aplo Flow pipettor system deck and toggle the ‘Reset tips to first position’ to appropriate position.
6. Click ‘Continue’ in the dialog box.
7.  Note that as the protocol is running, the button corresponding to the chip being processed will change colors and now function as a pause protocol button.

• Take care if vortexing, flicking, or spinning down the LNPs. This can result in a fragmented appearance when imaging.
• Only remove LNPs from storage and dilute immediately before adding to the reagent block.
• When diluting LNPs, we recommend performing two dilution steps if diluting over 1:2000 (for example, mix 1:1000 then 1:10 to perform a 1:10,000 dilution).
• When mixing LNP dilutions, we recommend mixing with 50% of the total solution volume. For large dilutions (i.e., 1:500 or higher), mix slowly 10 times by pipetting up and down. For small dilutions, mix at least 3 times by pipetting up and down. The Aplo Flow system will mix the samples again before adding them to the Assay Chip.