Processing human frontal cortex brain tissue for population-scale Oxford Nanopore long-read DNA sequencing SOP

Part 1: Brain Tissue Cutting (~2.5 hours for 16 samples) )

Add dry ice to a ice bucket

Place supplies (sterile weigh boat, razor blade, labeled empty 2mL protein LoBind microcentrifuge tubes and cooling block) in dry ice and allow to chill for ~ 0h 5m 0s

Obtain tissue samples from -80°Cfreezer and place in dry ice

Wear all necessary protective equipment (lab coat, face shield, double gloved) and complete the following steps within a chemical fume hood

Weigh labeled empty tube to tare scale, ensuring tube is centered

Working in the chemical fume hood, place the chilled weighing boat on top of the cooling tissue (use right hand to grip blade and cut tissue), use left hand just above, in between tissue and in front of hood, to shield any flying pieces)

Using the chilled razor blade, gently lift the cut tissue piece and transfer to the chilled labeled empty tube and weigh immediately, place back in dry ice immediately to avoid tissue thawing

Add or remove tissue using the method outlined above as required by input specifications ~40mg for frontal cortex, DNA recovery varies based on amount of gray matter vs. white matter)

Dispose of used weighing boat in a burn box and razor blade in the sharps waste container between each sample to prevent inter-sample contamination and keep all tissue samples on dry ice when not in use

Part 2: TissueRuptor Brain Tissue Disruption (~3 hours)

Place 15mL round tubes and cold Buffer CT on ice, chill centrifuge to4°C, and warm ThermoMixer to 55°C

Add 20µL of Proteinase K to the previous pellet

Add 50µL1X TE pH 8

Add 60µL Buffer CS

Add 100µL Buffer CLE3 and pipette mix 15X with a wide bore P200 pipette

Incubate for 1h 0m 0s on a ThermoMixer at 55°Cand 900 rpm

Spin on a mini-centrifuge for 0h 0m 2s  to remove liquid from the cap

Add 20µL of RNaseA and pipette mix 3X with a wide bore P200 pipette

Incubate for 0h 30m 0son a ThermoMixer at 55°C and 900 rpm

Spin the tube on a mini-centrifuge for0h 0m 2s to remove liquid from the cap

Add 50µL Buffer SB and vortex for0h 0m 10s at maximum speed (transfer any foam that appears)

Transfer brain tissue from previous steps to 15mL round tubes (keep on ice during the entire disruption process)

Add 750µL of cold Buffer CT

Submerge TissueRuptor probe tip in buffer and blend at max speed for 0h 0m 10s(place probe tip off to side to be cleaned later)

Transfer homogenate to a 2 mL Protein LoBind microcentrifuge tube including all undisrupted tissue chunks and any foam that forms

Pellet homogenate by centrifuging at 6,000 x g and 4°C for 0h 5m 0s. Discard supernatant (pellet may not be visible, so pipette carefully and avoid pipetting from the bottom of tube)

Add 1mLof cold Buffer CT and pipette mix 10X with a wide bore P1000 pipette to resuspend tissue

Pellet homogenate by centrifuging at 6,000 x g and 4°C for 0h 2m 0s. Discard supernatant (pellet may not be visible, so pipette carefully and avoid pipetting from the bottom of tube)

Pulse vortex for 0h 0m 1sx 5 times (max setting) to dislodge pellet

Part 3: KingFisher Apex Nanobind Tissue Big DNA protocol (~2 hours)

Prepare KingFisher Apex plates as follows:

Plate 1 - Lysis Binding: Sample + 50µL BL3

Plate 2 - Nanobind Storage: one 3mm Nanobind disk

Plate 3 - CW1 Wash 1: 600µLBuffer CW1

Plate 4 - CW1 Wash 2: 600µL Buffer CW1

Plate 5 - CW2 Wash 1: 600µL Buffer CW2

Plate 6 - CW2 Wash 2: 600µL Buffer CW2

Plate 7 - Elution: 100µL Buffer EB

Plate 8 - Tip: KingFisher Flex 96-Tip Combo

Run KingFisher Apex program “210804_nanobind_tissue_kf_apex_v2.kfx” (KF script available by request from Circulomics Inc)

After 0h 12m 0s when the program pauses, add 300µLIPA

Collect DNA by transferring eluate to a new 1.5 mL microcentrifuge tube OR if sample is to be sheared, the sample can be transferred to a DNA Fluid+ tube.

Let the sample rest at room temperature overnight to allow DNA to solubilize

Part 4: DNA Shearing (8 hours per 48 samples)

Hand shear DNA

Hand-shear 10X with 1mL Luer-Lock syringes and 1.5” needles (bringing sample up into needle and depressing plunger counts as 1 cycle)

Quantify:

1. Quantify the samples with the nanodrop and Qubit and size using the Agilent Tapestation 4200
2. Upload the tapestation reports
3. Add the quantifications
4. Only take forward samples that  are > 4.5ug. If a sample does not reach this requirement then repeat DNA extraction from Part 1.

Megaruptor 3 Shear with DNAFluid+  kit:

1. Transfer all of sample to DNA Fluid+ tubes if not in already
2. Make sample up to 150µL with nuclease free water
3. Attach the DNA Fluid+ needle onto the tube and push the entire item into the Megaruptor 3 slots until it fits snugly. If running fewer than 8 samples, put the tubes in the 1st and/or 8th slots, working your way in. Samples should always be balanced, if running an odd number of samples, samples can be balanced with an empty corresponding tube. Shear at speed 45 (takes around ~1h 0m 0s)
4. Once the MR3 shearing is finished, repeat the run by navigating back to the main menu and select speed 45 again (takes around ~1h 0m 0s)
5. Avoid any vortexing of DNA from this point on to avoid any unnecessary further shearing, instead mix by gently flicking the tube and spin down

Part 5: DNA Quantification

Quantify the samples with the nanodrop and Qubit and size using the Agilent Tapestation 4200

Upload the tapestation reports and and quantifications

For Step 6 the starting material must be > 4.5ug. If a sample does not reach this requirement then repeat DNA extraction from Part 1. 

DNA can be stored at 4° C for up to four weeks, or -80°Cindefinitely

Part 6: Library Prep (~6 hours, not including flushing and returning cells) )

A. DNA Repair and End-Prep

1. Put all the necessary reagents on ice to thaw and the Agencourt AMPure XP beads out at room temperature

2. Prepare the following in a 0.2 mL thin-walled PCR tube:

   • 48µL DNA (load 4.5 ug, this will likely be over 48 μL but that is fine, just adjust the amount of beads to match the total volume of this mixture)

   -3.5µL NEBNext FFPE DNA Repair Buffer (vortex)

   • 3.5µL Ultra II End-prep reaction buffer (vortex)

   • 3µL Ultra II End-prep enzyme mix (do not vortex)

   • 2µLNEBNext FFPE DNA Repair Mix (do not vortex)

3. Mix thoroughly by gently flicking tube or pipetting up and down 10X, and then spin down

4. Using a thermal cycler, incubate samples at 20°C for0h 5m 0s and 65°C

for0h 5m 0s

• Start and pause thermal cycler to allow lid to come t85°C before putting samples in

5. Allow Thermocycler to cool to 4°C and then remove your samples.

6. Resuspend the AMPure XP beads by vortexing

7. Transfer DNA samples to clean 1.5 mL Eppendorf DNA LoBind tube

8. Add60µL(or equivalent volume, see step 2) of resuspended beads to the reaction and mix by pipetting up and down 10X

9. Incubate for 0h 5m 0s at room temperature

10. Prepare 500µL per sample of fresh 75% ethanol in Nuclease-free water

11. Pellet sample on magnet until eluate is clear and colorless, about 0h 2m 0s

12. Pipette off the supernatant and retain, just in case the following quant is uncharacteristically or surprisingly low

13. With the samples remaining on the magnet, wash the beads with200µL of the ethanol, pipetting on the opposite wall (the goal here is to make sure the beads are fully covered) and making sure not to disturb the pellet, count to 3 and remove and discard ethanol

14. Repeat previous step

15. Spin down and place the tube back on magnet, pipetting off any residual ethanol

16. Allow to dry for ~0h 0m 30s but do not over-dry

17. Remove the tube from the magnetic rack and resuspend the pellet in 61µLNuclease-free water, incubate for 0h 3m 0s at room temperature (hold in hands and gently flick every so often)

18. Pellet the samples on a magnet until eluate is clear and colorless

19. Remove and retain61µL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube

20. The sample concentration must be > 40ng/ul. If the sample does not reach this requirement restart from Part 6.

21. It is possible to store samples at4°C overnight at this step if needed

B. Adapter Ligation and Clean-Up

1. Spin down the AMX-F, Quick T4 ligase, and LNB, then return to ice

• Do not allow AMX-F to remain at room temperature for too long

2. Thaw LNB at RT and mix by pipetting up and down (vortexing is ineffective due to viscosity)

3. Thaw EB at RT, mix by vortexing, spin down, and place on ice

4. Thaw SFB at RT, mix by vortexing, spin down, and keep at RT

5. In a 1.5 mL Eppendorf DNA LoBind tube, mix the following in order:

   • 60µLDNA sample (if not 60, add water until it is)

   • 25µL LNB

   • 10µL Quick T4

   -5µLAMX-F

6. Mix gently by flicking the tube and spin down

7. Incubate the reaction for 0h 10m 0sat RT

8. During this time, put flow cells out at RT

9. Resuspend beads by vortexing

10. Add 45µL of resuspended beads to the reaction and mix by flicking

11. Incubate for 0h 5m 0sat RT

12. Spin down sample and pellet on magnet

13. Pipette off the supernatant and retain, just in case the final elution quant is uncharacteristically or surprisingly low

14. Wash the beads with 250µL SFB, flick to resuspend and repellent, remove and discard supernatant

15. Repeat previous step

16. Spin down and place the tube back on magnet, pipetting off any residual SFB

17. Allow to dry for ~0h 0m 30s, but do not over-dry

18. Remove the tube from magnet and resuspend pellet in 25 μL EB, spin down, and incubate for 0h 20m 0s at 37°C

19. During this time, QC the flow cells (only use flow cells with >6000 pores)

20. Pellet the beads on magnet until eluate is clear and colorless

21. Remove and retain 25µL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube (this is the DNA library)

22. Quantify samples on Qubit

23. Reprep library  from Part  6 if < 1200 ng. 

24. Keep libraries on ice until ready to load on flow cell

C. Priming and Loading Flow Cell

1. Thaw SBII, FLT, and FB, vortex, and spin down

2. Thaw LBII

3. Add 30µL of thawed and mixed FLT directly to tube of FB and vortex

4. Expose inlet port on flow cell and draw back a small volume to remove any air bubbles (usually about 20-30 μL, just until a small volume of buffer enters the pipette tip)

5. Flush 500µL of Priming Mix into the inlet port of the flow cell, being extremely careful to avoid the introduction of air bubbles at the end

6. Wait 0h 5m 0s

7. During this time, separate the DNA library into three equal aliquots (ideally with 400 ng of DNA each). Bring each aliquot up to 24µL with EB. (i.e, if your final elution is exactly 1200 ng in 24 ul, move 8µLto three separate tubes and add 16µLof EB to each.)

8. Prepare the first library  mix for loading:

   • 75µL SBII (vortex)

   • 51µL LB (pipette up and down immediately before use)

   • 24µLDNA library in EB (400 ng)

9. Immediately load all 150µLof the library mix

10. Close valve to seal inlet port and close PromethION lid

11. Wait 0h 10m 0sand then initiate sequencing

12. Ideally, the library quants yielded at least 1200 ng to allow for 3X 400 ng loads, the latter 2 loaded approximately after 24 and 48 hours. However this will vary slightly depending on pore usage, data generated, as well as other factors i.e. if after 24 hours there are still +3000 pores then the sample does not need to be reloaded until 48 hours.

    - To wash and reload a flow cell, begin by thawing Wash Mix (WMX) on ice and Wash Diluent (DIL) at RT (DIL should be vortexed, WMX should NOT be vortexed, only spun)
    
    - Add`2µL`WMX into `398µL`DIL and pipette mix
    
    
    
    -Pause the PromethION runs and export .pdf reports
    
    -Rotate the inlet port cover to reveal inlet port 1
    
    -Using a P1000, insert tip into inlet port and draw back a small volume using the wheel to remove any air (usually around 20-30 μL)
    
     -Load `400µL`Flow Cell Wash Mix into the inlet port, avoiding any introduction of air
    
    
    
     -Wait `1h 0m 0s`
    
    
    
    -Repeat priming steps and reload samples (steps 1 - 12)
    

D. Flushing and Recycling Flow Cells (~15 minutes per set of 4 flow cells)

1. Following the completion of the sequencing, flow cells may be removed from the sequencer

2. Place enough absorbent material to take up approximately 4 mL of flush waste

3. Rotate valve to reveal inlet port 1

4. Place flow cell at a 45° angle on the absorbent material and, using a P1000, flush 1mL of DI water into the inlet port

5. Repeat 3 more times for a total of 4mL

6. Once complete, close the input port cover and remove all liquid from the waste port

7. Dispose of absorbent material as local biological waste guidelines dictate

8. Return flow cells to clear plastic tray in which it was shipped, making sure to record the flow cell IDs

9. Seal the tray with the sticker provided in the packaging

10. Put the clear plastic lid back on the tray

11. Place the tray back in the packaging

12. Place packaged cells in the returns box (large box can hold 80)

13. Once returns box is filled, follow the instructions here and follow the prompts to request the box to be sent back to Nanopore

1. Following 72 hours of sequencing the sample should yield an N50 ≥ 30kb with a data output ~ 100-160GB.

#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
https://lh6.googleusercontent.com/0xryslPCmkGicbTEKEzYakeiLrt4TpN5_BbIQE-zCnz3LowEsyyo29nlu0Eb48eGsg5jTkDxkmNKfNY4IEXwv2hss9GhK4HR9mx3hIX81yJ7xQjEcaFEZ6lhJe6E0BrCDqJjAWav