NHE Jan 12 Notes

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Nick Elder

Group 2 intestines

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.