TITLE: Protocol for 6mA labeling and HMW DNA extraction from fresh frozen human brain samples
AUTHORS: Jonas  Demeulemeester , Koen Theunis

[DESCRIPTION]
This protocol details the procedure of 6mA labeling and HMW DNA extraction of fresh frozen brain tissue. The protocol is inspired by Fiber-seq.
 


[STEPS]
SECTION: 6mA labeling and HMW DNA extraction
1. Place the Dounce homogenizer and pestles on ice, chill the centrifuge to 4 °C and preheat the ThermoMixer to 37 °C.
SECTION: 6mA labeling and HMW DNA extraction
2. Carefully transfer 3-4 (  diameter) tissue punch biopsies (~25 mg) to the chilled Dounce homogenizer.
 

SECTION: 6mA labeling and HMW DNA extraction
3. Add 500 µL of the 1X Lysis buffer and let the tissue thaw for 1 min.
SECTION: 6mA labeling and HMW DNA extraction
4. Gently homogenize the tissue 10X with pestle A and 10X with B.
SECTION: 6mA labeling and HMW DNA extraction
4.1. Push the tissue with the pestle firmly into the bottom of the Dounce chamber with each stroke (Down + Up = 1X).
SECTION: 6mA labeling and HMW DNA extraction
4.2. Keep the tissue between tip of pestle and the bottom of the Dounce chamber for thorough homogenization.
SECTION: 6mA labeling and HMW DNA extraction
4.3. Homogenate may become foamy, but this is not a cause for concern.
 

SECTION: 6mA labeling and HMW DNA extraction
5. Incubate on ice for 5 min before adding 1000 µL of 1X Wash buffer.
SECTION: 6mA labeling and HMW DNA extraction
6. Transfer the lysate to a 2 mL Protein LoBind microcentrifuge tube.
SECTION: 6mA labeling and HMW DNA extraction
7. Rinse the pestles and homogenizer with the remaining 500 µL 1X Wash buffer and add to the sample.
SECTION: 6mA labeling and HMW DNA extraction
8. Pellet homogenate by centrifuging at 700 x g and 4 °C for 5 min. Discard supernatant.
SECTION: 6mA labeling and HMW DNA extraction
9. Resuspend the pellet in 200 µL of 1X Hia5 labeling buffer – use a 1 mL or wide bore tip.
SECTION: 6mA labeling and HMW DNA extraction
10. Incubate on a ThermoMixer at 37 °C and 900 rpm for 30 min.

SECTION: 6mA labeling and HMW DNA extraction
11. Continue with Circulomics CBB Tissue protocol from step 8 onwards.
Continue with NEB Monarch HMW.

SECTION: Nanopore sequencing (LSK-110, PromethION)
12. According to ONT protocol Genomic DNA by Ligation (SQK-LSK110) with the following modifications:
SECTION: Nanopore sequencing (LSK-110, PromethION)
12.1. Start with 3 µg-4 µg of HMW DNA in 150 uL and sheer 25x with a 26G needle or in Megaruptor to 35kb.

SECTION: Nanopore sequencing (LSK-110, PromethION)
12.2. Adjust volumes end-prep and FFPE repair step accordingly (i.e. vol x 3), omit control strand (CS).
SECTION: Nanopore sequencing (LSK-110, PromethION)
12.4. Extend ligation step to 60 min at Room temperature.

SECTION: Nanopore sequencing (LSK-110, PromethION)
12.3. Extend end-prep and FFPE repair steps from 5 min to 30 min (i.e. 30min at 20 °C and 30 min at 65 °C).

SECTION: Nanopore sequencing (LSK-110, PromethION)
12.5. Elute the AMPure cleanups for 10 min and 20 min after the end-prep and ligation steps.

SECTION: Nanopore sequencing (LSK-110, PromethION)
12.6. This should yield a ~3x library, aim to load near the high end of the 5-50fmol range, typically ~8 µL.