Jan 12

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.