Prior to experimentation, the probe should be coated (including microchannel) and filled with mineral oil. For the coating procedure, a plasma cleaner, chemical hood, the FluidFM Coating Kit, SC Biopsy Kit and an oven that can be heated to 60°C are required (see Experimental Preparation in Feasibility of Biopsy Collection).

For sample collection, an RNAse-free work environment is necessary to preserve RNA integrity. For tips on how to clean your workstation see (see RNA Handling Guidelines in Clean Biopsy Collection). A microcentrifuge and 4°C refrigerator for sample storage, is also required.

Which steps does the biopsy procedure have?

The procedure has 12 steps, which are listed in the Extraction workflow in the ARYA software which guides the user sequentially through them:

1. Enter preload buffer:
   A 1 µL droplet of preload buffer is placed on the droplet slide in the biopsy plate. The probe then approaches the droplet, and the tip of the cantilever enters the droplet, without immersing the whole consumable.
   Tip: Preload buffer helps prevent RNA degradation and initiates protein lysis as soon as the extraction occurs.
   
   
2. Aspirate buffer:
   Some of the preload buffer (approx. 1-2 pL) is aspirated into the cantilever by applying a negative pressure.
   
   
3. Go to Sample:
   The cantilever is taken out of the droplet and the probe is moved into the well with the experimental cells.
   
   
4. Align Probe: 
   The probe approaches an empty space on the surface of the dish, pressing against it at a set force, and prompting the user to align the probe again for greater accuracy when piercing the cell.
   
   
5. Extraction:
   The probe gently approaches the selected cell using the force-feedback control, pierces it and extracts the contents by applying a negative pressure. 
   
   
6. Water wash:
   The probe, containing the preload buffer and cell contents, is dipped in water three times to remove any cell media that may be on the cantilever or consumable. 
   
   
7. Load droplet:
   A 1 µL droplet of collection buffer is placed on the droplet slide in the biopsy plate. 
   
   
8. Dry probe:
   The probe needs to be dried in order to be able to controllably enter the deposition droplet, without the deposition droplet adhering to the consumable and mixing with droplets already on the consumable. is dried by bringing the sides of the consumable into contact with the Watman paper that is mounted in the biopsy plate. This absorbs the water droplet from the consumable and cantilever.
   
   
9. Enter deposition buffer: 
   The probe then approaches the droplet of collection buffer, and the tip of the cantilever enters the droplet, without immersing the whole consumable.
   
   
10. Dispense extract:
    The combination of preload buffer and cell extract is deposited into the droplet by applying a positive pressure.
    
    
11. Rinse cantilever:
    Negative and positive pressures are applied in the cantilever to aspirate collection buffer and inject it back into the droplet respectively, effectively rinsing the cantilever.
    
    
12. Remove droplet:
    The collection buffer droplet containing the preload and cell extract is removed from the droplet slide using a pipette.

From here, the droplet is pipetted into a collection tube, where the sample is spun down using a centrifuge, labelled and placed in the fridge to await library preparation and sequencing

The cells’ locations are automatically saved on the system and can be used in the ‘Observation’ workflow. This workflow can be used to track the cells’ movements over a certain period of time, so that the cell can be relocated and rebiopsied later, if desired.

How is the sample taken from the cell analysed?

Once the droplet containing the cell extract has been collected and stored, the sample undergoes single-cell RNA sequencing analysis.

The analysis begins with library preparation, where the extracted RNA is converted into a sequenceable format. Live-seq samples contain ultra-low input volumes (typically 1-5 picoliters of cellular content), requiring specialised library preparation methods that can work with minimal starting material while preserving the integrity of the RNA information.

Following library preparation, the samples undergo high-throughput sequencing to identify and quantify the genes that were active in the biopsied cell. This shows which genes were expressed by the cell and at what levels, providing insights into the cell's functional state, identity, and response to experimental conditions.

What do I need to conduct a library preparation and sequencing?

For optimal results with Live-seq samples, a high-sensitivity library preparation method that can be used with ultra-low input samples is required. We recommend using Lexogen's LUTHOR HD (High-Definition Single-Cell 3' mRNA-Seq Library Prep Kit), which is specifically designed for such samples like those obtained through Live-seq extractions with inputs as low as 1-10 pg of RNA. This is best complemented by Lexogen's 12 nt UDI (Unique Dual Indexing) system for optimal read rescue capabilities.

The procedure also requires a thermal cycler for PCR amplification, a magnetic bead separation system, a microcentrifuge and pipettes with low-binding tips. Like the biopsy procedure, both the environment and consumables must be RNase-free. A -80°C freezer, -20°C freezer and 4°C refrigerator are necessary for sample and reagent storage. Finally, access to an Illumina sequencing platform is necessary for sequencing.

Which steps do library preparation have?

Library Preparation Steps (using LUTHOR HD):

1. THOR amplification: Oligo(dT) Primers containing Unique Molecular Identifier (UMI), an Illumina-compatible P7 linker and a T7 promotor sequence prime reverse transcription of polyadenylated RNAs, focusing on the 3’ end of transcripts.  A linear amplification of the RNA strands is reached through subsequent in vitro transcription
   
   
2. Library generation: Libraries are generated through reverse transcription using “Random Displacement Stop primers” containing Illumina-compatible P5 linker.
   
   
3. Library amplification: During PCR, Lexogen’s Unique Dual Indices (UDI) are added which enables multiplexing and sequencing of the libraries.
   
   
4. Quality control: The prepared libraries are assessed for concentration, size distribution and quality before sequencing.
   
   

The entire process from library preparation to sequencing results typically takes 7 days, enabling rapid analysis of the temporal gene expression changes captured through Live-seq's unique live-cell biopsy approach.

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