Week of 10/21/24-10/25/24

Intro

This week was slightly shortened as the lab was closed on Friday, 10/25. What we did do this week was start off with prepping samples for desiccation and put them into the desiccation chamber to sit over the course of the weekend. Samples were pulled out on Friday by Chad as he had access to the lab and removed the samples so they weren't too desiccated to be able to perform RNA isolation on them. This does mean, however, that the samples were allowed to sit for longer than normal total time.

Methods

Pre-desiccation cell packing 

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

Plates were moved into desiccator after 48 hours of drying time

Results & Discussion

During this week we also fully went through the sequencing procedure in detail, counting how many tubes and of what kind we would need to be fully prepared. Each section was broken down by the procedure itself, along with what reagents it would require, then we went through and triple checked our tube counts before rounding up slightly for error. From there we started setting aside materials we would need to be autoclaved so we would be prepared for when we finally start sequencing. 

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.

Conclusion

Next week we will complete the RNA extraction on the desiccated cells and, based on the results of that as well as the following quality checks, we may or may not be able to move forward with the sequencing. 

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