Difference between revisions of "DM Notes 1.12.16"
(Created page with "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 ti...") |
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| − | RNAseq protocol | + | RNAseq protocol |
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| − | -RNA fragmentation, cDNA synthesis, and clean-up | + | 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? | |
| − | + | ||
| − | + | 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) | ||
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| + | Product: | ||
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| + | Look at images in pptx | ||
| + | |||
| + | Second band of images: gel purified RNA | ||
| + | |||
| + | Third band of images: reamplified (considered good cDNA library for RNAseq) | ||
Revision as of 20:41, 27 January 2016
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)
