Apr 04, 2025

Public workspaceProtocol for 6mA labeling and HMW DNA extraction from fresh frozen human brain samples

DOI
dx.doi.org/10.17504/protocols.io.j8nlkw54wl5r/v1
  • 1Laboratory of Integrative Cancer Genomics, VIB-KULeuven;
  • 2Laboratory of Computational Biology, VIB-KULeuven
  • Aertslab
Koen Theunis
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkw54wl5r/v1
Protocol Citation: Jonas Demeulemeester , Koen Theunis 2025. Protocol for 6mA labeling and HMW DNA extraction from fresh frozen human brain samples. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw54wl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 09, 2022
Last Modified: April 04, 2025
Protocol Integer ID: 69773
Keywords: HMW DNA extraction, Nanopore sequencing, PromethION, ASAPCRN
Abstract
This protocol details the procedure of 6mA labeling and HMW DNA extraction of fresh frozen brain tissue. The protocol is inspired by Fiber-seq.
CITATION
Stergachis AB, Debo BM, Haugen E, Churchman LS, Stamatoyannopoulos JA (2020). Single-molecule regulatory architectures captured by chromatin fiber sequencing.. Science (New York, N.Y.).
LINK
https://doi.org/10.1126/science.aaz1646

Attachments
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Materials
Prepare buffers (Volumes above are indicated per sample):

10x stocks of Wash buffer base (no spermidine/Tween) and Labeling buffer base (no spermidine/SAM/Hia5).

2 mL Wash buffer -> take Amount488 µL and add Digitonin + 50XProtInh to make Lysis buffer.
Amount50 µL labeling buffer / 250.000 cells (typically ~ 1E6 cells -> Amount200 µL ).

Make Concentration25 millimolar (mM) spermidine stocks fresh monthly and store at Temperature-20 °C .
Add Amount2 µL spermidine (liquid, ~Concentration6.38 Molarity (M) ) to Amount350 µL Concentration0.1 Molarity (M) HCl and Amount160 µL H2O – check pH with strips!


ABCD
10X Wash/Lysis base (10X-WLB) (200uL / sample)
StockFinal for 10XV (uL)
Tris-HCl pH 7.41000 mM100 mM100
NaCl5000 mM100 mM20
Water880
Total1000
ABCD
10X Hia5 labeling base (10X-H5B) (20uL / sample)
StockFinal for 10XV (uL)
Tris-HCl pH 81000 mM150 mM150
NaCl5000 mM150 mM30
KCl1000 mM600 mM600
EDTA pH 8500 mM10 mM20
EDTA pH 8250 mM5 mM20
Water180
Total1000
ABCDE
1X Wash buffer
StockFinalV (uL)X5
10X WLB10 X1 X2301150
BSA10%0.10%23115
Spermidine pH 7.425 mM0.5 mM46230
Tween-2010%0.10%23115
Water19789890
Total230011500
ABCDE
1X Lysis buffer
StockFinalV (uL)X5
Wash buffer4882440
Digitonin5%0.02%210
ProteaseInh50 X1 X1050
Total5002500
ABCDE
1X Hia5 labeling buffer
StockFinalV (uL)X5
10X-H5B10 X1 X20100
BSA100.1210
Spermidine pH 7.4250.5420
SAM320.8525
Hia5 enzyme2505420
Water165825
Total2001000
6mA labeling and HMW DNA extraction
6mA labeling and HMW DNA extraction
Place the Dounce homogenizer and pestles TemperatureOn ice , chill the centrifuge to Temperature4 °C and preheat the ThermoMixer to Temperature37 °C .
Carefully transfer 3-4 (Thikness2 mm diameter) tissue punch biopsies (~Amount25 mg ) to the chilled Dounce homogenizer.
Note
Keep the Dounce homogenizer on ice during the entire disruption process.

Add Amount500 µL of the 1X Lysis buffer and let the tissue thaw for Duration00:01:00 .
1m
Pipetting
Gently homogenize the tissue 10X with pestle A and 10X with B.
Push the tissue with the pestle firmly into the bottom of the Dounce chamber with each stroke (Down + Up = 1X).
Keep the tissue between tip of pestle and the bottom of the Dounce chamber for thorough homogenization.
Homogenate may become foamy, but this is not a cause for concern.
Note
In the next step, transfer any foam that forms.

Incubate TemperatureOn ice for Duration00:05:00 before adding Amount1000 µL of 1X Wash buffer.
5m
Incubation
Pipetting
Transfer the lysate to a 2 mL Protein LoBind microcentrifuge tube.
Pipetting
Rinse the pestles and homogenizer with the remaining Amount500 µL 1X Wash buffer and add to the sample.
Pipetting
Wash
Pellet homogenate by centrifuging at Centrifigation700 x g and Temperature4 °C for Duration00:05:00 . Discard supernatant.
5m
Centrifigation
Resuspend the pellet in Amount200 µL of 1X Hia5 labeling buffer – use a Amount1 mL or wide bore tip.
Incubate on a ThermoMixer at Temperature37 °C and Centrifigation900 rpm for Duration00:30:00 .

30m
Incubation
  • Continue with Circulomics CBB Tissue protocol from step 8 onwards.
  • Continue with NEB Monarch HMW.

Nanopore sequencing (LSK-110, PromethION)
Nanopore sequencing (LSK-110, PromethION)
2h 5m
2h 5m
According to ONT protocol Genomic DNA by Ligation (SQK-LSK110) with the following modifications:
Start with Amount3 µg -Amount4 µg of HMW DNA in 150 uL and sheer 25x with a 26G needle or in Megaruptor to 35kb.

Adjust volumes end-prep and FFPE repair step accordingly (i.e. vol x 3), omit control strand (CS).
Extend end-prep and FFPE repair steps from Duration00:05:00 to Duration00:30:00 (i.e. 30min at Temperature20 °C and 30 min at Temperature65 °C ).

35m
Extend ligation step to Duration01:00:00 at TemperatureRoom temperature .

1h
Elute the AMPure cleanups for Duration00:10:00 and Duration00:20:00 after the end-prep and ligation steps.

30m
This should yield a ~3x library, aim to load near the high end of the 5-50fmol range, typically ~Amount8 µL .
Note
Note 1:Library prep yield is typically 30-50%.
Note 2:The amount loaded can be reduced during subsequent flushes to balance seq yield with # flushes.

Citations
Stergachis AB, Debo BM, Haugen E, Churchman LS, Stamatoyannopoulos JA. Single-molecule regulatory architectures captured by chromatin fiber sequencing.
https://doi.org/10.1126/science.aaz1646