Introduction

This protocol describes a method to extract genomic DNA from human brain tissue using the QIAGEN MagAttract HMW DNA Kit and can be automated by using the KingFisher Flex or Duo Prime systems using the MagAttract handbook for these systems.

This extraction method recovers ~8-15 µg of DNA from 25 mg of human brain tissue. Sequencing libraries were prepared using the Ligation Sequencing Kit both with and without a high throughput DNA shearing step using the FastPrep-96, before sequencing on PromethION Flow Cells. Flow cell output was increased by washing the flow cell using the Flow Cell Wash Kit and re-loading prepared library.

Materials

• 25 mg of frozen brain tissue
• Ligation Sequencing Kit
• QIAGEN MagAttract HWM DNA Kit
• Scalpel
• Weighing boats
• 1 ml 96-well plates
• Plate sealer (e.g. Bio-Rad PX1 PCR plate sealer)
• Pierceable Foil Heat Seals
• 2 ml DNA LoBind Eppendorf Tubes
• P1000 pipette and tips
• P1000 wide-bore tips
• Weighing scales capable of measuring 25 mg
• Thermomixer
• Magnetic rack
• FastPrep (e.g. FastPrep-96 Classic bead beating grinder and lysis system)
  

Optional materials

• QIAGEN TissueRuptor II
• 15 ml Falcon Tube
• Agilent Femto Pulse System
• Femto Pulse System DNA Analysis Kit (e.g. Genomic DNA 165 kb Kit)

Method

Tissue lysis

Brain tissue should always be kept frozen prior to processing the sample. Please note, when the frozen sample is sliced, it will thaw quickly and generate condensation around the tissue, which will affect the weight. Please take the necessary precautions depending on the type and origin of the tissues you are working with.

1. Use a scalpel to thinly slice 25 mg of frozen brain tissue and place in a sterile weighing boat.
2. Homogenise the tissue to ensure adequate penetration of the lysis buffer into the tissue to accelerate the lysis step. This can be performed in one of two ways:
   • Recommended: Use a scalpel to mince the brain tissue in a weighing boat until it turns into a paste-like consistency which should take several minutes. Transfer the minced tissue into a 2 ml DNA LoBind Eppendorf Tube or 1 ml 96-well plate. Add 400 µl of MagAttract Lysis buffer (ATL) (MagAttract HMW DNA Kit) and 40 µl of proteinase K to the sample immediately. To ensure even dispersion through the sample, slowly mix by pipetting, using a P1000 pipette and standard P1000 tip approximately 5 times until the tissue is homogenised in the solution.
   • Alternative (using TissueRuptor): Place 25 mg of sliced tissue in a 15 ml Falcon Tube and add 400 µl of MagAttract Lysis buffer (ATL). Shear the sample for 2 seconds at minimum speed in the TissueRuptor. Add 40 µl of proteinase K to the sample.
3. Place the tubes or 96-well plate in a thermomixer preheated to 56°C and shake at 1000 rpm for 1 hour.
4. Remove the samples from the thermomixer.

Note: The tissue should be completely or almost completely dissolved. If not, it is possible the tissue was not properly broken down in the previous step. To dissolve any intact tissue, pipette mix the tissue using a P1000 pipette tip to break up any tissue aggregates and incubate for another 30-60 minutes. If some small pieces of tissue are still visible in solution after the lysis incubation, precipitate these by centrifuging at 16,000 x g for 3 minutes and remove any supernatant without disturbing the pellet. This is important as any undissolved pieces of tissue may bind to the MagAttract beads which can be very difficult to remove. 5. Take forward the homogenate to extract the DNA using a modified version of the QIAGEN MagAttract protocol.

DNA extraction

Note: This is a modified version of the QIAGEN Magattract HMW DNA protocol.

1. Add 8 µl of RNase and mix gently by flicking the tube.
2. Incubate at room temperature for 2 minutes.
3. Add 300 µl of AL buffer and mix using a P1000 wide-bore pipette 5-10 times.
4. Add 560 µl of MB buffer followed by 60 µl of MagAttract beads. Thoroughly resuspend by vortexing for 2-3 seconds. If using a 96-well plate, cover with adhesive film or use a plate sealer.
5. Incubate in a thermomixer for 3 minutes at 1000 rpm at room temperature.
6. Place the plate/tube on a magnet to pellet the magnetic beads. Remove the supernatant by pipetting without disturbing the bead pellet.
7. Add 700 µl of MW1 and resuspend the beads.
8. Use a thermomixer to mix the reaction for 2 minutes at 1000 rpm at room temperature.
9. Place the plate/tube on a magnet to pellet the magnetic beads. Remove the supernatant by pipetting without disturbing the bead pellet.
10. Repeat steps 7, 8 and 9 to complete a second wash with MW1.
11. Pre-warm Buffer AE to 80°C.
12. Add 700 µl of Buffer PE to the bead pellet.
13. Use a thermomixer to mix the reaction for 2 minutes at 1000 rpm at room temperature.
14. Place the plate/tube on a magnet to pellet the magnetic beads. Remove the supernatant by pipetting without disturbing the bead pellet.
15. Repeat steps 12, 13 and 14 to complete a second wash with Buffer PE.
16. Resuspend the beads with 150-200 µl Buffer AE, which has been pre-heated to 80°C, to elute the DNA.
17. Use a thermomixer to mix for 1 hour at 600 rpm at 37°C.
18. Place the plate/tube on a magnet to pellet the magnetic beads. Retain the supernatant containing the eluted DNA.
19. Quantify the recovered DNA using a Qubit fluorometer.

DNA shearing

1. After recovering and quantifying the elution from the MagAttract beads, transfer the eluate into a new 1 ml 96-well plate. It is important to keep the volumes used in this shearing method constant as different volumes and different plates may have an impact on shearing performance. It is also advisable to avoid the formation of bubbles when transferring the DNA sample as bubbles may effect the shearing and impact the final output.

Note: We recommend the plates are thoroughly sealed using a heat sealer and appropriate seal. 2. Place the sealed plate in the FastPrep-96. 3. Shear at 1200 rpm for 1 minute. 4. Remove the plate and spin down to make sure no liquid is left in the seal. 5. Optional: Evaluate the shearing by analysing an aliquot before and after shearing using an Agilent Femto Pulse (or equivalent for QC). 6. After the extraction and shearing step, proceed with the library preparation protocol for the Ligation Sequencing Kit.

Results

Figure 1. a) Read length analysis, b) output analysis and c) Femto Pulse analysis of sheared and unsheared gDNA extracted from human brain samples.

Version	Change
V3, August 2023	Updated URL addresses
V2, July 2022	Reformatted images
V1, July 2022	Initial publication