RNA library preparation part 1

Sara was very kind to take the time out of her schedule to show me the library preparation ropes! We were working through this protocol. I will need to finish up the protocol once I figure out what tagging primers I need!

Notes

• Library preparation for samples extracted March 31: 25-117, 25-058, 25-193, 5-124, 5-187
• Calculated the volume of RNA to use from each sample to standardize adding 250 ng of RNA to each sample here
  • 25-058: 4.3 µL RNA, 45.7 µL NF H₂O
  • 25-193: 6.7 µL RNA, 43.3 µL NF H₂O
  • 5-124: 3.2 µL RNA, 46.8 µL NF H₂O
  • 5-187: 3.2 µL RNA, 46.8 µL NF H₂O
  • 25-117: 2.8 µL RNA, 47.2 µL NF H₂O
• Picked up some tips from Sara about using a multichannel pipet!
  • Rock the tips onto the pipet when loading
  • When pipetting up small volumes, tilt the tips away from the beads and stick them onto the bottom. Once the tips touch the bottom, keep the tips at the bottom and rock them back and forth to suck up the liquid.
  • When pipetting up large volumes, start sucking up the liquid with the pipet tips angled as they move down into the bottom of the tubes/plate. This prevents the liquid from overflowing.
• Some tips working with beads
  • NEVER LET THE BEADS DRY!
  • Generally, keep the pipet tips angled away from the beads
  • When washing the beads, it helps to pipet the liquid back onto the beads to ensure they are hydrated and mixed back into the liquid.
• Finished the poly-A selection and cDNA synthesis and placed libraries in the -20ºC

Going forward

1. Find all RNA samples and move to -20ºC
2. Move extracted RNA to the -80ºC
3. Extract RNA
4. Find all metabolomics samples
5. Send samples for metabolomics
6. Try NEB UltraExpress Kit
7. Order library preparation materials
8. Examine HOBO data from 2023 experiment
9. Demographic data analysis for 2023 paper
10. Start methods and results of 2023 paper