Long-read sequencing offers a powerful tool for studying complex transcriptomes, as demonstrated by Dr. Martijn Kerkhofs. By capturing full-length RNA and DNA molecules, long-read sequencing reveals isoform diversity and alternative splicing that are often missed by short-read methods. This approach provides deeper insights into gene expression and regulation, making it invaluable for research in fields like neuroscience.

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1.   Why use this method?

Long-read sequencing, such as that enabled by Oxford Nanopore Technologies (ONT), provides crucial insights into the full-length structure of DNA, c DNA and RNA molecules. Unlike short-read methods, Oxford Nanopore Technologies captures complete transcripts, preserving isoform diversity. This method is particularly powerful for studying complex transcriptomes, like those of the somatosensory cortex, where alternative splicing and isoform regulation play significant roles in neuronal function and development. The ability to sequence entire molecules allows for a detailed understanding of gene expression dynamics and the identification of previously unannotated isoforms, contributing to advances in neuroscience research. This method is especially valuable in neuroscience for profiling the complex transcriptomes of brain regions.

2. What you’ll need

Biological Material:

• Somatosensory cortex samples: Fresh or frozen tissue, prepared in an RNase-free environment to maintain RNA integrity

Consumables:

• RNase-free pipette tips
• RNase -free tubes
• Spin columns with silica membranes

Reagents:

• Lytic buffer
• DNase (if isolating RNA)
• RNase (if isolating DNA)
• Purifying buffer
• Washing buffer
• Elution buffer
• Ethanol (for washing steps)
• Reverse transcription reagents
• ONT for library preparation kits and flow cells

Equipment:

• Tissue homogenizer or mortar and pestle (for tissue disruption)
• Centrifuge
• Nanopore sequencing device (e.g., MinION, GridION, or PromethION)
• Thermocycler
• Spectrophotometer or Nanodrop (for RNA purity and quantity assessment)
• Bioanalyzer or TapeStation (for RNA integrity check)

3. Step-by-step instructions

1. Acquire biological material (the quality of the starting material will determine the quality of the output at the end) through micro-dissection of the somato-sensory cortex
2. Isolate RNA (or DNA)- depending on DNA or RNA, you add a RNAse or DNAse step, respectively

DNA/RNA Sample

• Input: starting material, such as cells, tissues, or other biological samples containing RNA
• The goal is to extract RNA (and sometimes DNA) for downstream applications

Lysis with Lytic Buffer

• Action: Add the lytic buffer to the sample
• Purpose: This breaks open the cells, releasing nucleic acids (RNA/DNA) into solution
• After mixing, the lysate is ready for purification

Binding on Spin Column

• Action: The lysate is transferred to a spin column containing a silica membrane, which selectively binds RNA/DNA under specific buffer conditions
• Centrifuge: Spinning forces the sample through the column, and RNA/DNA binds to the silica surface, while impurities pass through

Purifying Buffer

• Action: A purifying buffer is applied to the column
• Purpose: This removes proteins, salts, and other contaminants from the bound nucleic acids
• After centrifugation, these impurities are discarded in the flow-through

Washing Buffer

• Action: Apply washing buffer to the column
• Purpose: This step ensures any residual contaminants (e.g., salts, ethanol) are removed completely
• Centrifugation is repeated to clear out the wash solution

Dry Spin

• Action: Perform an additional spin without any added buffer
• Purpose: This removes any remaining ethanol from the washing buffer, preventing interference with downstream applications

Elution with Elution Buffer

• Action: Add elution buffer to the spin column
• Purpose: The buffer releases the RNA/DNA from the silica membrane into the solution
• Centrifuge: Spinning the column collects the purified RNA/DNA in a clean tube below

Eluted DNA/RNA

• Output: The final purified RNA/DNA is collected in the eluate and is ready for downstream applications

3. Quality Control -Spectrophotometer

260/280 ratio RNA 260/280 ratio = 2.0: indicates pure RNA. DNA 260/280 ratio = 1.8: indicates pure DNA

260/230 ratio this ratio indicates contamination from substances like salts, carbohydrates, or phenol, which absorb at 230 nm, a high 260/230 ratio suggests minimal impurities.

4. RNA Integrity Number (RIN) ratio 18S/28S ribosomal RNA using Bioanalyzer or TapeStation. Library preparation

Library preparation= the process whereby the input (RNA or DNA) is modified to suit the sequencing method

• Reverse Transcription
• Adapter ligation
• Formation of Y-junctions
• Addition of motor proteins

5. Sequencing (Nanopore sequencing)

DNA, or cDNA is unwound by the motor protein, and one strand is translocated through the pore. Each base gives a characteristic change in the ionic current, allowing the sequence of bases to be inferred

6. Bioinformatic Analysis

4. Practical tips

• Ensure RNA is of high quality to maximize sequencing performance
• Regularly calibrate instruments to ensure accurate measurements
• Run pilot experiments to optimize library preparation conditions

5. Critical appraisal & implications for future research

While Oxford Nanopore Technologies long-read sequencing is a powerful tool for transcriptome analysis several limitations exist

• Base-calling accuracy is lower compared to short-read technologies

Future improvements in nanopore chemistry and bioinformatics tools will enhance accuracy and usability Integrating Oxford Nanopore Technologies sequencing with complementary methods such as short-read sequencing can provide more comprehensive insights into transcriptome complexity.

^{This protocol is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license, allowing sharing and adaptation for non-commercial purposes with proper attribution.}