meth_3.2.4 PureLink RNA Mini Kit modified for sponges Marine Microbes Coastal Project

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AM Project ID: Legacy

  1. Cut all three pieces of each specimen (n=3) into small pieces under liquid nitrogen. Mix the small pieces for each specimen, but don’t mix pieces of the sponge replicates.
  2. Add approximately 0.5 g of material to a tube separately for each sponge replicate.
  3. Add 1 mL of Trizol directly in the tube (still frozen) and let it defrost while cutting the tissue with small scissors
  4. Transfer the liquid (with the crushed tissue) into a tube with beads and beat for 30 s at 5.5 speed
  5. Follow the protocol for Trizol extraction (i.e., PureLink RNA Mini Kit) with the DNase I treatment step, elute in 100 µL. (This usually results in 700 – 1000 ng/µL of total RNA by Qubit)
  6. Check the samples by agarose gel and Bioanalyzer before continuing
  7. Deplete the polyA + sequences with “Poly(A) purist Kit” following the protocol, but discarding the beads and recover the RNA from the supernatant with the PureLink RNA Mini Kit doing another DNase I treatment step
  8. Check the samples with universal bacterial 16S rRNA gene primers (to confirm that there is no contaminant DNA)
  9. Use this RNA with another round of with “Poly(A) purist Kit” as before (DNA I treatment can be omitted, if previous PCR was negative)
  10. Use 5 µg of the previous RNA (PolyA-) to deplete in ribosomal RNA using “RiboZero Bacteria Kit” following the protocol.
  11. Remove the beads and precipitate the supernatant with the ethanol method (adding 2 µL Glycogen, 1/10 volume 3 M NaOAc, 2.5 volumes of ethanol and incubate overnight at -20 °C).
  12. Resuspend the final pellet in water. The final RNA should be labelled according to the instruction given in the spreadsheet “sampling_schedule” and stored at -80 °C.