RNA Extraction and Sonora Sequencing

 Introduction: 

This week included an RNA extraction from D. aquaticus with the intent of using this as template for qPCR primer validations, and another attempt at sequencing of D. sonorensis. The experiment with D. aquaticus has been founded upon the sequencing data produced from this lab, identifying the Type III-A CRISPR-Cas system that is fairly unique within the genus, and especially being the only system in the species without interaction or even the presence of another type. The original project was actually utilizing D. geothermalis, but its multisystem components were a potential source of major error or a level of interaction we were not prepared to investigate yet. By studying D. aquaticus, we have effectively decreased the level of complexity for the time being and likely have decreased the range in temperature that will be used for the final experiment. The genes of interest in this species is Cas1 and Cas10, both major factors in bacterial immunity. Cas1 is prominent for its ability to detect viral offenders, and Cas10 is the primary "cutting" protein in the Type III-A complex that is responsible for quenching the viral threats. Ultimately, these proteins will be analyzed in their transcription according to a change in temperature, both hot and cold in relativity to the optimal growth temperature of 30 degrees Celsius. Before this part of the experiment can be carried out, we must first determine what is cold or hot to D. aquaticus, by conducting qPCR experiments comparing the transcription of cold shock and heat shock proteins between 30 degrees Celsius and our designated hot and cold temperatures. By detecting a significant increase the transcription of each shock protein at experimentally determined temperatures, we can confidently use these temperatures for the primary experiment thereafter, knowing the cells are experiencing a shock. There are then six sets of primers to be validated prior to this step taking place, and the first set of primers validated were CSP (cold shock protein). 

Methods:

For sequencing of D. sonorensis, the dilution factor of the sample was reduced to more formally align with the suggestion from Illumina support. An approximate 1:2 dilution was done with 9 ul of sample being well mixed into 11 ul of 0.1X TE buffer. The 20 ul of sample was then loaded into the apparatus containing the flow cell and ran for about 18 hours. 

RNA extraction of D. aquaticus was carried out with one broth of 10 ml. The culture was gram stained for cleanliness, and Nanodrop value of an OD600 at 1.63. All remaining culture was divided into two tubes and centrifuged to build a pellet of approximately 5 ml each. The ThermoFisher RNA isolation kit was utilized with some changes to the standard protocol. Firstly, the cells underwent bead beating for a total of 10 minutes, with 1 min on/1 min off and on ice. The DNase step was also completed, though listed as optional in the protocol. For elution, PCR water was used at 25 ul volume and allowed to incubate in the column at ambient temperature for about 5 minutes prior to centrifugation. The samples were tested on the Nanodrop for concentration and purity values. 

The validation of CSP primers were done using the Luna OneStep qPCR standard protocol, with an initial input of 88 ng of undiluted RNA for tube one. Then serial 1:5 dilutions of the first tube were done to tube 5 with a final dilution of 1:625. Each dilution was done in triplicate, and briefly analyzed for approximate success. 

Results:

The RNA samples isolated in the week were both clean, with each having a set of values well over 2.0 for the 260/280 and the 260/230. Each of their concentrations were also well over 1 ug/ul with 1.8 ug/ul in Tube 1 and 2.4 ug/ul in Tube 2. 

The sequencing of D. sonorensis was actually completed this second attempt, but the data is still unclear whether contamination was present and if the data will then be useful. Currently a 23% estimation of the genome is acquired. 

Finally, the qPCR results from the primer validation of CSP was successful, with each dilution on a log scale being spaced evenly from each other and the first detection seen at cycle 14. The data has not been validated by equation yet, but is conditionally finished. 

RNA Sample

Concentration (ng/ul)

260/280

260/230

1

1,800

2.08

2.25

2

2,400

2.12

2.32


Discussion:

The primer set of CSP will need to be confirmed by technology, but it appears one set of primers from the six has now been validated and a protocol for primer validation been set. The results were so visibly good that it was decided it should be done the same from then on. The next set should be our heat shock protein, and then the reference genes and Cas1 and Cas10 will be completed as well. The sequencing of D. sonorensis is still undetermined, but the structure of its genome is proving to be formidable. The short read sequencing of Illumina may not be suitable for the abnormalities that is sonora, and long read nanopore ONT technology may be the savior by being able to overcome this by being less affected by the genome structures. By using increased throughput and long read, we may be able to hybridize a sequence that can be very usable for future endeavors. 



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