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| | '''January 12, 2016''' | | '''January 12, 2016''' |
| − | RNAseq protocol:
| + | [[Notes]] |
| − | Flash freeze tissue from six snakes, two organs per snake
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| − | RNA range: 100 ng to 10 micrograms
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| − | --> how do we know that the correct portion of tissue was sampled?
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| − | Convert RNA to mRNA using polyT oligonucleotides attached to beads, such that mRNA base pairs with beads
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| − | Convert mRNA to cDNA with RT (reverse transcriptase, uses RNA as a template to make DNA) and dNTP)
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| − | | |
| − | -RNA fragmentation, cDNA synthesis, and clean-up
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| − | high throughput sequencing has only ~75bp of accurate reads
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| − | fragmentation takes one long mRNA and cuts it into similarly sized fragments so that there are more edges to read from
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| − | How to prime every RNA simultaneously? Generate every possible hexamer for the primers (4^6 different combinations)
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| − | Product:
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| − | Look at images in pptx
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| − | Second band of images: gel purified RNA
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| − | Third band of images: reamplified (considered good cDNA library for RNAseq)
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