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.