Reviewing Sequencing Procedure

 We started the week off strong with a pre-desiccation procedure, then assisting Evan with a procedure, and finally going over our sequencing protocol. 

We did pre-desiccation on our A B and C flasks grown last week, following the usual cell packing procedure and then moved them to the egg the following day. Friday they were removed from the vacuum as it was unable to hold pressure that long but we were unable to perform RNA iso, so they dehydrated over the weekend. 

1. Normalize 3ml of culture to an OD between 0.95-1.00

2. Spin down 1ml of media, remove supernatant and resuspend pellet in nuclease free water

3. Resuspend pellet, then centrifuge and remove supernatant

4. Add the second ml of culture, repeat washing steps

5. Add the third ml of culture, repeat washing steps

6. Plate 100ul dots into 1 inch kapton squares in a 6 well plate in triplicate

We were also able to help Evan with a twitch motility procedure, preparing the specialized plates with the unique molds and later helping to inoculate the center of each plate with p81 before leaving it to incubate. 

Twitch motility is a form of surface translocation exhibited by certain bacteria that possess pili, which are thin, hair-like appendages extending from the bacterial cell surface. These pili function through a dynamic cycle of extension, attachment to a surface, and retraction, which allows the bacteria to "walk" across solid surfaces in a jerky, intermittent manner. The mechanism involves complex protein machinery that assembles, extends, and retracts the pili, with proteins like PilB (for extension) and PilT (for retraction) generating the characteristic twitching movement.

Twitch motility assays are experimental techniques used to visualize and quantify this movement, typically performed on semi-solid agar surfaces or using microscopic observation methods. Researchers commonly employ techniques such as stab inoculation assays, where bacteria are inserted into soft agar, and the spreading pattern is observed, or direct microscopic tracking using phase-contrast or fluorescence microscopy to capture the rapid extension and retraction of pili. 

We were also able to discuss our sequencing procedure which is the cDNA-PCR Sequencing V14 (SQK-PCS114) from ONT. We went over each section and broke down the procedure as well as calculated the amount and type of materials needed for each section.

The cDNA-PCR Sequencing V14 (SQK-PCS114) kit is a specialized workflow designed for preparing complementary DNA (cDNA) libraries for nanopore sequencing, particularly suited for RNA-based studies. The workflow can be divided into several critical stages, each each helping to transform RNA samples into sequencable nanopore libraries. The primary goal of this method is to convert RNA molecules into a format compatible with nanopore sequencing technology while preserving the original RNA sequence information.

The kit has the following sections:

1. RNA Extraction and Quality Control: In this initial phase, high-quality RNA is isolated from the biological sample. Researchers must ensure RNA integrity and purity, as these factors significantly impact downstream sequencing performance. The extracted RNA undergoes quality assessment using techniques like gel electrophoresis or spectrophotometric measurements to confirm its suitability for the cDNA conversion process.
2. Reverse Transcription and First-Strand cDNA Synthesis: During this stage, RNA is converted into complementary DNA (cDNA) using reverse transcriptase enzymes. This process creates a DNA copy of the original RNA transcript, which is more stable and amenable to subsequent amplification and sequencing steps. The reverse transcription step often incorporates specific primers or adapters that will later facilitate library preparation and sequencing.
3. Second-Strand cDNA Synthesis: After the first-strand cDNA is generated, a complementary second strand is synthesized. This double-stranded cDNA provides a more robust template for subsequent library preparation and helps improve the overall representation of the original RNA population.
4. PCR Amplification: The cDNA is then amplified using polymerase chain reaction (PCR). This step serves multiple crucial functions: it increases the quantity of starting material, helps normalize the representation of different transcripts, and introduces sequencing adapters necessary for nanopore library preparation. The PCR step is carefully optimized to minimize potential biases and maintain the relative abundance of different RNA transcripts.
5. End Repair and Adapter Ligation: In this section, the amplified cDNA undergoes end repair to create blunt-ended fragments and then has specific nanopore sequencing adapters ligated to its ends. These adapters are critical for several reasons: they provide priming sites for sequencing, enable electrical detection during nanopore sequencing, and help orient the DNA molecules as they pass through the nanopore.
6. Library Preparation and Cleanup: The prepared library is then purified to remove excess adapters, enzymes, and other reaction components. This cleanup step ensures that only the desired cDNA molecules with properly ligated adapters are retained for sequencing.
7. Sequencing Preparation: Finally, the library is prepared for loading onto a nanopore flow cell. This involves adjusting the library's concentration and preparing it in a format compatible with the nanopore sequencing instrument. The prepared library will then pass through protein nanopores, generating electrical signals that can be translated back into nucleotide sequences.

The cDNA-PCR Sequencing V14 kit is particularly advantageous for studying RNA populations, including messenger RNA (mRNA), microRNA, and other RNA species. It offers flexibility in handling various sample types and RNA quantities, making it valuable for research in areas like gene expression analysis, transcriptomics, and molecular biology. Like all sequencing kits, it requires careful execution at each stage to maintain the fidelity and representativeness of the original RNA sample. Proper controls, optimization, and attention to detail are crucial for obtaining high-quality sequencing results.