DM Notes 1.12.16

RNAseq protocol

Flash freeze tissue from six snakes, two organs per snake

RNA range: 100 ng to 10 micrograms

--> how do we know that the correct portion of tissue was sampled?

Convert RNA to mRNA using polyT oligonucleotides attached to beads, such that mRNA base pairs with beads

Convert mRNA to cDNA with RT (reverse transcriptase, uses RNA as a template to make DNA) and dNTP)

-RNA fragmentation, cDNA synthesis, and clean-up

high throughput sequencing has only ~75bp of accurate reads

fragmentation takes one long mRNA and cuts it into similarly sized fragments so that there are more edges to read from

How to prime every RNA simultaneously? Generate every possible hexamer for the primers (4^6 different combinations)

Product:

Look at images in pptx

Second band of images: gel purified RNA

Third band of images: reamplified (considered good cDNA library for RNAseq)

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