meth_3.2.3 PureLink RNA Mini Kit modified for seagrass Marine Microbes Coastal Project

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Extract RNA from seagrass leaves using PureLink RNA Mini Kit as per the manufacturer’s protocol (Life Technologies) with the addition of On-column PureLink DNase (Life Technologies) to remove contaminating gDNA.

RNA clean

  1. In PCR plate, pipette from aliquots 100 μL of RNAclean solution. (Gently shake before to resuspend beads)
  2. Add ~50 µL sample and pipette to mix *Mix gently as fast pipetting can displace sample from well.
  3. Leave to stand for a minimum of 10 min to allow strands to bind to magnet beads.
  4. Transfer PCR plate to SuperMagnet. Leave for 10 min for rings to form.
  5. Carefully remove solution in wells without disturbing the ring of beads.
  6. Add 200 μL 70 % ethanol and leave for 30 s.
  7. Careful not to disturb the bead ring, pipette mix the ethanol to wash and remove from sample. Repeat for a total of 3 washes.
  8. After the last wash, make sure all ethanol is pipetted out from wells.
  9. Allow to air dry for 9-10 min. Do NOT allow beads to dry!
  10. Remove plate from magnet. Add 40 μL of DEPC RNase free water for RNA samples and pipette to elute beads and strands. Elution is rapid.
  11. Place plate back on magnet for 10 min.
  12. Carefully pipette out the sample into 1.5 µL sterile tubes prepared earlier and place on ice.
  13. If you suspect carryover of magnet beads, place tube back on magnet plate and being aliquoting 10 μL RNA to remaining 1.5 µL tubes. With care, a final drop of sample containing magnet beads can be separated and removed.
  14. Place 3 x 10 µL RNA aliquots into -80 °C freezer. Keep one “dirty” sample on ice to nanodrop. Labelled according to the scheme outlined in the spreadsheet “sample_schedule”.

RNA quality control, transcriptome and metatranscriptome sequencing

  1. Check RNA quantity and quality using Nanochip technology (Agilent 2100 Bioanalyzer) according to manufacturer’s instructions (Agilent).
  2. Store high-quality RNA samples (integrity number >7) in -80 °C freezer for further sequencing.
  3. Send RNA samples by sequencing (5GB per sample).