Once the cells have been onboarded and at least one feasibility experiment on both the cells and buffer has been completed, the clean biopsy collection can begin. This involves collecting biopsies from cells in an RNase-free environment and preparing them for storage until library preparation.

Note: Cytosurge recommends the Lexogen LUTHOR HD (High-Definition Single-Cell 3' mRNA-Seq Library Prep Kit), (Lexogen UDI 12 nt Unique Dual Indexing Sets | Lexogen) for library preparation, as this kit is sensitive enough to detect the low quantities of RNA that are present in a single cell biopsy. If a different kit is to be used in your clean collection, it is recommended to first carry out a feasibility test by running a library preparation and sequencing experiment on the RNA controls listed above to ensure that the kit is sensitive enough to detect low levels of RNA.

Experimental Preparation

The probe coating, biopsy plate preparation and wash plate preparation should be carried out as described previously in the feasibility experiment (Experimental Preparation in the Feasibility of Biopsy Collection). Additionally, certain precautions will need to be taken in order to ensure an RNase-free environment.

Note: Sources of RNase contamination on lab surfaces often include bacterial and fungal spores and dead cells shed from human skin (e.g., hands).

RNA Handling Guidelines

To avoid RNA degradation, the environment needs to be devoid of ribonucleases, therefore we recommend following these guidelines:

• Protect the samples from yourself:
• • Wear fresh gloves and a clean lab coat. Be careful not to touch your face or hair with gloved hands.
  • Change gloves frequently; especially if they touch anything outside of the RNase-free working area.
  • Wear a face mask to prevent contamination from breathing.
  • Do not talk above the sample or the RNase-free working area.
    
    
• Work in RNase-free conditions, with surfaces cleaned using RNase Zap or RNase Away:
• • Handle RNA-containing samples only in a designated RNase-free area which has been thoroughly cleaned with commercially available RNase inactivating agents (RNase Zap or RNase Away).
  • Metal parts inside the instrument are NOT RNase Zap nor RNase Away compatible, however can be cleaned with 70% ethanol and/or UV irradiated instead.
  • RNase Zap or RNase Away needs to be rinsed off the surfaces with nuclease free water since it can damage the RNA in the sample. Wait for at least 2 minutes before rinsing off the surfaces
    
    
• Use RNase-free consumables:
• • Use RNase-free reagents (e.g. RNase-free water)
  • Use RNA handling dedicated consumables and plasticware (e.g. pipette tips, Eppendorf tubes)
  • UV can also be used to kill RNases. UV all reagent containers, consumable containers, pipettes, the biopsy plate and components before beginning the experiment. The biopsy plate and the components are also compatible with the RNase Zap or RNase Away.
  • When working, keep reagent tubes and bottles closed whenever possible.
• When carrying out the sample collections: 
• • Use RNase inhibitors in the collection buffer for storage of biopsies.
  • Carry out the biopsy workflow at room temperature (23°C, temperature controller off) to reduce RNA degradation.
  • Keep the collection buffer and any RNA-containing samples on ice whenever possible.
  • Store the biopsies at 4°C if being processed within the next five working days, or at –80°C if being processed at a later date or being shipped.

If the lab has expertise in RNA handling, please refer to the internal protocols that complement these recommendations.

Experimental Procedure

Sample Storage

For the clean collection, positive controls, negative controls and biopsy samples that are to be processed for library preparation within following five working days can be stored at 4°C. Samples that are to be processed at a later time, or being shipped, should be immediately frozen at -20°C and transferred to -80°C at the end of the collection.

Out-of-OMNIUM Controls

Before beginning the experiment, consideration should be given to which negative and positive controls should be used and their quantity. Negative controls can be created using RNase-free water, while positive controls can be created using different quantities of universal human reference RNA (UHRR). Positive controls can also be created using different quantities of RNA from the specific cell line being used in the biopsy experiment for more powerful analysis. We recommend using at least one negative control and three positive controls:

• 0 pg (negative)
• 0.1 pg (positive)
• 1 pg (positive)
• 10 pg (positive)

The 1 pg control should be the closest in gene quantity to the cell biopsies. Controls can also be created in duplicates or even triplicates for greater accuracy.

Tip: Positive controls at such low RNA concentrations are highly sensitive to pipetting techniques. Ensure to familiarise yourself with best-practice pipetting techniques for low-input serial dilution before beginning.

Note: Cytosurge recommends the Lexogen LUTHOR HD (High-Definition Single-Cell 3' mRNA-Seq Library Prep Kit), (Lexogen UDI 12 nt Unique Dual Indexing Sets | Lexogen) for library preparation, as this kit is sensitive enough to detect the low quantities of RNA that are present in a single cell biopsy. If a different kit is to be used in your clean collection, it is recommended to first carry out a feasibility test by running a library preparation and sequencing experiment on the RNA controls listed above to ensure that the kit is sensitive enough to detect low levels of RNA.

In-OMNIUM Controls

It is also possible to take in-OMNIUM negative controls to ensure no contamination is introduced to the samples by the probe or the droplet slide. We recommend taking the in-OMNIUM controls just before using a new probe to ensure it is free of contaminants. However, controls can also be taken after probe use or in between samples to ensure there is no carry-over.

In order to take in-OMNIUM controls, follow these steps (ensure the probe is dry before beginning):

1. Pipette 5.2 µL of collection buffer into a collection tube on ice.
2. Pipette a 1 µL droplet of collection buffer from the collection tube onto an unused spot on the droplet slide.
3. Approach and enter the droplet as described previously (Preload Droplet Entry in Buffer Testing in Experimental Procedure in Feasibility of Biopsy Collection)
4. Rinse the cantilever by aspirating and depositing 5 pL of the buffer at least 5 times. Make sure the FluidFM Nanosyringe only contains oil before moving to the next step.
5. Remove the droplet slide from the OMNIUM.
6. Pipette the droplet from the droplet slide back into the collection tube. Optionally, rinse the spot on the droplet slide with 1 µL of collection buffer from the same collection tube. This should also be returned to the collection tube afterwards.
7. Spin down the collection tube for a couple of seconds and label before storage.

Biopsies

In order to extract clean biopsies from your cells, follow the steps (ensure the probe is dry before beginning):

1. Pipette 6.2 µL of collection buffer into a collection tube on ice.
2. Pipette a 1 µL droplet of collection buffer from the collection tube onto an unused spot on the droplet slide.
3. Approach and enter the droplet as described previously (Preload Droplet Entry in Buffer Testing in Experimental Procedure in Feasibility of Biopsy Collection).
4. Aspirate the preload buffer as described previously (Aspirate Preload Buffer in Buffer Testing in Experimental Procedure in Feasibility of Biopsy Collection).
5. Navigate to the cell location using the ‘Go to Sample’ step.
6. Align the probe as described previously (Probe Alignment in Define Piercing Parameters in Experimental Procedure in Feasibility of Biopsy Collection).
7. Extract a biopsy from the cell as described previously (Extraction in Define Piercing Parameters in Experimental Procedure in Feasibility of Biopsy Collection).
8. Wash the probe: Configure the wash to run the water wash as described in Wash Plate Configuration (Wash Plate Preparation in Experimental Preparation in the Feasibility of Biopsy Collection).
9. Load the collection droplet by pipetting another 1 µL droplet of collection buffer from the collection tube onto an unused spot on the droplet slide. The next steps should be carried out as quickly as possible to avoid evaporation of the droplet.
10. Dry the probe as described previously (Probe Washing section of Buffer Testing in Experimental Procedure in Feasibility of Biopsy Collection).
11. Deposit the extract as described previously (Extract Deposition section of Buffer Testing in Experimental Procedure in Feasibility of Biopsy Collection).
12. Rinse the probe as described previously (Rinsing section of Buffer Testing in Experimental Procedure in Feasibility of Biopsy Collection).
13. Remove the droplet slide from the OMNIUM.
14. Pipette the droplet, containing the cell extract, from the droplet slide back into the collection tube. Optionally, rinse the spot on the droplet slide with 1 µL of collection buffer from the same collection tube. This should also be returned to the collection tube afterwards.
15. Spin down the collection tube for a couple of seconds and label before storage.
16. Following collection, the collection volume of each sample can be determined using the volume analysis tool (Volume Tool in Feasibility of Biopsy Viability).