JP Jan 12 16
Notes: (RNA seq protocol pdf + For Davidson pptx)
Don’t want to give RNA time to degrade. Tubes, -40C baths. Protocol : .1 g of tissue = 100micrograms Shave off tissue, RNA isolation kit. How do we know the right part of the organ was sampled? only desired organ harvested? Room for Error
1. Poly[A] enrichment
used a poly-T oligonucleotides (since mRNA has a string of AAAAs) attached to beads (this isolates mRNA from other RNA
2. RNA fragmentation
only about 75 bases can be read. 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.
Don’t sequence RNA ; use cDNA (complementary DNA) (transcribed the mRNA) Reverse Transcriptase enzyme; uses RNA as template to make DNA
Primer: how to prime every cDNA (with infinite combo of sequences) ACACTCTTTCCCTACACGACGCTCTTCCGATCTNxxxNNNNNN Hexamer with every possible combination to attach to the cDNA. Temps cool enough so that every attachment will occur when it can. Every possible sequence accounted for in this situation.
3. cut out a certain molecular weight from gel purification; reamplify 500bp ; considered a good library (pptx)
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).
Do you know what you did and can you describe it?