NHE 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.
