Week of 10/7/24-10/11/24

Intro

This week consisted of inoculating 3 new flasks and 3 new plates from the freezebacks created recently. We started pre-desiccation procedures again and further discussed what sequencing would look like and what kind of results we should expect to see/interpret.

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

We discussed more RNA sequencing and what exactly that process would look like. It begins with RNA extraction and is followed by multiple quality checks to ensure a lack of degradation. Next, purification focuses on isolating mRNA through methods like polyA selection and rRNA depletion. Library prep transforms RNA into sequencing-compatible fragments by fragmenting the RNA, synthesizing cDNA, adding adapters, and performing PCR amplification. High- throughput sequencing generates millions of DNA sequence reads, which are then subjected to analysis. This portion includes quality control of the sequencing reads, as well as alignment to reference genomes or transcriptomes, read count quantification, normalization, and statistical analysis. The final stage includes interpretation of the data, which includes pathway analysis, functional annotation, and validation through alternative methodological approaches.  

Conclusion

Next week we will complete what should hopefully be our last round of RNA extraction before moving forward with sequencing.

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