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| − | http://www.bio.davidson.edu/courses/Bio343/LabMethods_2016.html
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| − | http://gcat.davidson.edu/mediawiki-1.19.1/index.php/Burmese_Python_RNAseq_Project
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| − | http://gcat.davidson.edu/mediawiki-1.19.1/index.php/Julia_Preziosi
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| − | Glossary of terms: http://gcat.davidson.edu/GcatWiki/index.php/Blueberry_Genome_Project_for_Bio343#A
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| − | Notes: (RNA seq protocol pdf + For Davidson pptx)
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| − | Don’t want to give RNA time to degrade. Tubes, -40C baths.
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| − | Protocol :
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| − | .1 g of tissue = 100micrograms
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| − | Shave off tissue, RNA isolation kit.
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| − | How do we know the right part of the organ was sampled? only desired organ harvested? Room for Error
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| − | 1. Poly[A] enrichment
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| − | used a poly-T oligonucleotides (since mRNA has a string of AAAAs) attached to beads (this isolates mRNA from other RNA
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| − | 2. RNA fragmentation
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| − | only about 75 bases can be read.
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| − | Advantage of fragmenting RNA before cDNA: randomly fragmented the long mRNA = can get a lot more reads, more edges to read 75 in from. Roughly the same size.
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| − | Don’t sequence RNA ; use cDNA (complementary DNA) (transcribed the mRNA)
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| − | Reverse Transcriptase enzyme; uses RNA as template to make DNA
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| − | Primer: how to prime every cDNA (with infinite combo of sequences)
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| − | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNxxxNNNNNN
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| − | Hexamer with every possible combination to attach to the cDNA.
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| − | Temps cool enough so that every attachment will occur when it can.
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| − | Every possible sequence accounted for in this situation.
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| − | 3.
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| − | cut out a certain molecular weight from gel purification; reamplify 500bp ; considered a good library (pptx)
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| − | Tasked to figure out if there’s a gene transcribed for the three fed snakes that’s not in the three unfed snakes, from the many 75bp results (which are disassembled).
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| − | Do you know what you did and can you describe it?
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