Jan 12 Notes

6 snakes 3 fed/3 unfed Flash-fozen in dry ice and ethanol to preserve RNA 0.1g samples of frozen tissue were shaved off -possibility for the tissue to not have been representative -yielded 100 ug RNA -need 10 ug for amplification Isolate only mRNA to use to create cDNA -beads used to isolate mRNA were covered in poly-T tails -beads were also magnetic -isolates 2% of RNA removed from bead and mixed with Reverse Transcriptase (RT), dNTP's, and made cDNA RNA fragmentation - normally want to avoid fragmenting RNA BUT: High-throughput sequencing only produces 75 bp reads Fragmentation allows for more coverage in sequencing Now need to prime RNA for sequencing BUT every the RNA has been fragmented Create a library of primers that contain bar code regions (3 nucleotides) and randomly generated hexameters Purified PCR product was run on a gel. Fragment of about 500 bp was excised and reamplified to confirm data. These fragments were then used to produce the RNA seq data.