Hot alkaline lysis gDNA extraction from formalin-fixed archival tissues

Visually assess the specimen. If the specimen appears decomposed, consider selecting an alternative specimen. Generally, specimens with discernible internal organs tend to have higher likelihood of success. If the internal organs have been removed, this may indicate that the specimen had begun to decompose prior to fixation and will be unsuitable for DNA sequencing. Consult with your curatorial team as, in some cases, removal of the internal organs may have been conducted for alternative purposes that may not have influenced DNA preservation.

From the jar containing your visually suitable specimen, take an aliquot of the specimen media into a 15 mL tube for down-stream measurement of residual formaldehyde and pH. If using a pH meter, you'll want to remove enough media such that you can adequately immerse the probe - typically at least 7 mL.

Measure the pH of the media.

Measure the residual formaldehyde concentration [F] of the media.

Rate your visually well-preserved specimen for suitability against your sequencing objectives.

• If the pH measures lower than 6 or the [F] measures greater than 10,000 mg/L, consider using an alternative specimen or limit your interrogation to small amplicon-based approaches.
• If the pH is neutral and the [F] sits above 0 and below 10,000 mg/L, treat the specimen as formalin-fixed and well-preserved, thus suitable for broader-scale DNA sequencing, depending on extraction yield.
• If the pH is neutral and there is no detectable formaldehyde, treat the specimen as ethanol-preserved.

Dissect at least 50 mg of your target tissue and remove to a 2 mL tubes containing 70% ethanol.

Transfer and store the dissected tissue at room temperature until further processing.

Pulverisation

Weigh and record the total tissue mass, subsampling to 50 mg if more tissue is available.

Note: More than 50 mg can be processed. Here we have limited it to 50 mg as we find this amount pulverises well without rupturing the tissueTUBE bag.

Transfer the tissue to an extra thick TT05 Covaris tissueTUBE bag and attach a 1 mL Covaris milliTUBE.

Dispense ~ 1L liquid nitrogen to a cryo dewar and flash freeze the tissueTUBE bag through immersion for approximately 30 sec.

Apply 2-3 level 6 impacts to the tissueTUBE with the Covaris CryoPrep, flash freezing the sample between impacts.

Tissue re-hydration and formaldehyde mop-up

Resuspend the tissue in cold 50% ETOH and transfer to a 15 mL tube using a wide-bore pipet tip. Rinse the bag with cold 50% ETOH to collect all material. Bring volume to 10 mL with cold 50% ETOH. Rock for 10 min at 4ºC.

Spin 10min at 750 x g 4ºC. Remove supernatant and resuspend tissue in 10 mL cold 30% ETOH. Rock for 10 min at 4ºC.

Spin 10min at 750 x g 4ºC. Remove supernatant and resuspend tissue in 10 mL cold milliQ water. Rock for 10 min at 4ºC.

Spin 10min at 750 x g 4ºC. Remove supernatant and resuspend tissue in 10 mL cold GTE buffer. Rock over night at 4ºC.

Spin the tube for 10 min at 750 x g 4ºC. Remove and discard all but 1 mL of the GTE buffer.

Resuspend the tissue in the 1 mL of retain GTE and transfer the tissue to a 2 mL screw-cap tube with a wide-bore pipet.

Spin the screw-cap tube for 10 min at 750 x g 4ºC. Remove the supernatant and add 500 μL Alkaline Lysis Buffer (0.1NaOH, pH 13; 1% SDS) to the pellet.

Secure the tube upright in an autoclave save container and autoclave at 120 ºC for 25 minutes.

Phenol:chloroform:isoamyl alcohol extraction

Add equal volume phenol:chloroform:isoamyl alcohol (25:24:1) to the screw-cap tube of lysate, briefly vortex to resuspend and then rock for 5 min at room temperature.

Centrifuge the tube at maximum speed in a microcentrifuge for 5 min at room temperature.

Remove the aqueous phase to a new 2 mL tube and add 100 μL 10 mM Tris-HCl pH 8.0 to the organic phase. Rock the tube containing the organic phase for an additional 5 min.

Centrifuge the organic phase tube at maximum speed in a microcentrifuge for 5 min at room temperature. Combine the aqueous phase from this extraction with that recovered from the first extraction. Discard organic phases in the appropriate waste stream.

OPTIONAL: Add 1 μL RNase A to the extract and incubate at room temperature for 30 min.

Repeat the phenol:chloroform:isoamyl alcohol extraction.

Chloroform clean-up

Add equal volume chloroform and rock for 5 min at room temperature.

Centrifuge the tube at maximum speed in a microcentrifuge for 5 min at room temperature. Remove the aqueous phase to a new 2 mL tube.

Repeat chloroform extraction until the phenol is completely removed. Usually, we only perform one chloroform extraction and trust that the phenol is removed if we were able to remove the aqueous phase cleanly.

DNA concentration with SPRI beads

To each tube, add 1.5X volume small fragment-optimised bead solution and incubate at room temperature for 15 minutes.

Briefly spin the tubes and place them on a magnet for 10 minutes or until the solution is clear.

Remove the supernatant and wash the bead pellet twice with fresh 70% ethanol.

Briefly spin the tubes and return to the magnet. Remove remaining ethanol with a small volume pipette and allow the beads to dry for 30 seconds.

Add 30 μL 10 mM Tris-HCl, pH 8.0 and resuspend the beads by pipetting up and down.

Incubate the tube at 37ºC for 15 minutes then pellet the beads on the magnet once again for 15 minutes.

Transfer the supernatant containing your extracted DNA to a new tube.

Measure the DNA concetration via Qubit and/or Tapestation.

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