TITLE: DiMeLo-seq: Directed Methylation with Long-read sequencing
AUTHORS: Nicolas  Altemose, Annie  Maslan, Owen  Smith, Kousik  Sundararajan, Rachel  Brown, Aaron  Straight, Aaron   Streets

[DESCRIPTION]
Directed Methylation and Long-read sequencing (DiMeLo-seq) is a powerful method to map protein-DNA interactions at a single-molecule level across the genome (including repetitive regions). It can be multiplexed to analyze multiple base modifications at once (e.g. endogenous CpG methylation and directed pA-Hia5 adenine methylation). Additionally, PCR amplification is not necessary for this protocol, which means that sequencing readout is proportional to protein-DNA interaction frequency. Finally, DiMeLo-seq can be used to map multiple protein interactions across a long single molecule. 



[STEPS]
SECTION: Introduction
1.  
This protocol is part of the broader Directed Methylation and Long-read sequencing (DiMeLo-seq) method. DiMeLo-seq targets a protein A-Hia5 methyltransferase fusion protein (pA-Hia5) to a protein of interest (using an antibody). Hia5 methylates adenines in the vicinity of the protein, the DNA is extracted, and long-read sequencing with modified base-calling is performed. Overall, this allows for the determination of protein location throughout the genome (including at repetitive regions) on a single-molecule level. This protocol is specifically used for the application of DiMeLo-seq in situ, on permeabilized nuclei. 

   

   

SECTION: Reagent Preparation
2. General Notes
Prepare all reagents fresh
Keep all reagents on ice
Filter all buffers through a 0.2 μm filter
SECTION: Reagent Preparation
2.1. Digitonin:

Solubilize digitonin in preheated 95ºC Milli-Q water to create a 5% digitonin solution (e.g. 10 mg/200 μl).
SECTION: Reagent Preparation
2.2. Wash Buffer:

Prepare wash buffer with the following amounts/concentrations.

 
 
 

SECTION: Reagent Preparation
2.3. Dig-Wash Buffer:

Add 0.02% digitonin to wash buffer. For example, add 20 μL of 5% digitonin solution to 5 mL wash buffer.
SECTION: Reagent Preparation
2.4. Tween-Wash Buffer:

Add 0.1% Tween-20 to wash buffer. For example, add 50 μL Tween-20 to 50 mL wash buffer. 
SECTION: Reagent Preparation
2.5. Activation Buffer:

Create the activation buffer, but wait to add SAM until the activation step.

 
 
 

SECTION: General Notes
3. 
All spins are at 4ºC for 3 minutes at 500 x g.
Spinning in a swinging bucket rotator can help pellet the nuclei.
To prevent nuclei from lining the side of the tube, break all spins into two parts: 2 minutes with the tube hinge facing inward, followed by 1 minute with the tube hinge facing outward.
Working with Eppendorf DNA LoBind tubes can reduce loss of material.
Use wide bore tips when working with nuclei.
Do not use NP-40 or Triton-X100, as they appear to dramatically reduce methylation activity.
The best digitonin concentration may vary by cell type. For HEK293T, GM12878, and HG002 cells, 0.02% works well. You can test different concentrations of digitonin and verify permeabilization and nuclear integrity by Trypan blue staining. For example, you may try 0.02% to 0.1% digitonin.
We use Tween to reduce hydrophilic non-specific interactions and BSA to reduce hydrophobic non-specific interactions. We’ve also found BSA at the activation step significantly increases methylation activity as well.
The best primary antibody concentration may vary by protein target of interest. A 1:50 dilution works well for targeting LMNB1, CTCF, and CENP-A, but this may need to be varied depending on target abundance and antibody specificity.
A secondary antibody binding step following primary antibody binding and before pA-Hia5 binding reduced total methylation and specificity. Including a secondary antibody binding step is not recommended.
SECTION: Optional Fixation
4. Fixation is optional - not necessary for completion of protocol.
SECTION: Optional Fixation
4.1. Resuspend cells in PBS.
SECTION: Optional Fixation
4.2. Add PFA to 0.1% (e.g. 6.2 µl of 16% PFA to 1 ml cells) for 2 minutes while gently vortexing.
SECTION: Optional Fixation
4.3. Add 1.25 M glycine (sterile; 0.938 g in 10 ml) to twice the molar concentration of PFA to stop the crosslinking (e.g. 60 µl of 1.25 M glycine to 1 ml).
SECTION: Optional Fixation
4.4. Centrifuge 3 minutes at 500 x g at 4°C and remove the supernatant.
SECTION: Optional Fixation
4.5. Resuspend the fixed cells in Dig-Wash buffer (Nuclear Isolation, step 5.3).
SECTION: Nuclear Isolation
5. Extraction and isolation of nuclei
SECTION: Nuclear Isolation
5.1. Prepare cells (1M-5M per condition).
SECTION: Nuclear Isolation
5.2. Wash cells in PBS. Spin and remove supernatant.
SECTION: Nuclear Isolation
5.3. Resuspend cells in 1 ml Dig-Wash buffer. Incubate for 5 minutes on ice.
SECTION: Nuclear Isolation
5.4. Split nuclei suspension into separate tubes for each condition.
SECTION: Nuclear Isolation
5.5. Spin and remove supernatant.
SECTION: Nuclear Isolation
5.6. Quality Control:

Check permeabilization was successful by taking 1 µl of the nuclei following the 5-minute incubation on ice, diluting to 10 µl with PBS, and staining with Trypan Blue. Alternatively, fix with 1% PFA for 2 minutes at room temperature and wash with Tween-Wash. Spin and remove supernatant. Resuspend in fluoromount with 1:500 Hoechst.

SECTION: Primary Antibody Binding
6. Bind the primary antibody targeted to your protein of interest.
SECTION: Primary Antibody Binding
6.1. Gently resolve each pellet in 200 µl Tween-Wash containing primary antibody at 1:50 or the optimal dilution for your antibody and target.
SECTION: Primary Antibody Binding
6.2. Place on rotator at 4°C for ~2 hr.
SECTION: Primary Antibody Binding
6.3. Spin and remove supernatant.
SECTION: Primary Antibody Binding
6.4. Wash twice with 0.95 ml Tween-Wash. For each wash, gently and completely resolve the pellet. This may take pipetting up and down ~10 times. Following resuspension, place on rotator at 4°C for 5 minutes before spinning down.
SECTION: Quantify Protein Concentration
7. Quantify concentration of pA-Hia5 for use in step 8 (pA-Hia5 binding).

SECTION: Quantify Protein Concentration
7.1. Thaw protein from -80°C at room temperature and then move to ice immediately.
SECTION: Quantify Protein Concentration
7.2. Spin at 4°C for 10 minutes at 10,000 x g or higher.
SECTION: Quantify Protein Concentration
7.3. Transfer the supernatant to a new tube and save it, discarding the previous tube .
SECTION: Quantify Protein Concentration
7.4. Use Qubit with 2 µl sample volume to quantify protein concentration.
SECTION: pA-Hia5 Binding
8. Bind pA-Hia5 to antibody. Protocol to express and purify the pA-Hia5 protein can be found on protocols.io under the title "pA-Hia5 Protein Expression and Purification". 
SECTION: pA-Hia5 Binding
8.1. Gently resolve pellet in 200 µl Tween-Wash containing 200 nM pA-Hia5. See protein quantification protocol above (Step 7). 
SECTION: pA-Hia5 Binding
8.2. Place on rotator at 4°C for 2 hrs.
SECTION: pA-Hia5 Binding
8.3. Spin and remove supernatant.
SECTION: pA-Hia5 Binding
8.4. Wash twice with 0.95 ml Tween-Wash. For each wash, gently and completely resolve the pellet. Following resuspension, place on rotator at 4°C for 5 minutes before spinning down.
SECTION: Quality Control
9. Quality control of steps completed so far, using a secondary antibody for immunofluorescence.

Note: Can fix first or last (just before mounting)
SECTION: Quality Control
9.1. Add 1.6 µl of 16% PFA to 25 µl of nuclei in Tween-Wash (taken from the 0.95 ml final wash) for 1% total PFA concentration.
SECTION: Quality Control
9.2. Incubate at room temperature for 5 minutes.
SECTION: Quality Control
9.3. Add 975 µl of Tween-Wash to stop the fixation by dilution.
SECTION: Quality Control
9.4. Add 1 µl secondary antibody.
SECTION: Quality Control
9.5. Put on rotator for 30 minutes at room temperature, protected from light.
SECTION: Quality Control
9.6. Wash 2 times (or just once). Pellet likely won’t be visible.
SECTION: Quality Control
9.7. Resuspend in mounting media after last wash. Use as little as possible, ideally 5 µl.
SECTION: Quality Control
9.8. Put 5 µl on a slide, make sure there are no bubbles, and put on a coverslip.
SECTION: Quality Control
9.9. Seal with nailpolish along the edges.
SECTION: Quality Control
9.10. Image or put at -20°C once the nail polish has dried.
SECTION: Activation
10. Activation of Hia5 MTase activity using S-adenosylmethionine (SAM).
SECTION: Activation
10.1. Gently resolve pellet in 100 µl of Activation Buffer per sample. Be sure to add SAM to 800 µM to the activation buffer at this step!
SECTION: Activation
10.2. Incubate at 37°C for 2 hrs. Replenish SAM by adding 800 µM at 1 hr. Pipet mix every 30 minutes. 
SECTION: Activation
10.3. Spin and remove supernatant. Alternatively, add proteinase K and RNase A directly to the activation reaction before column extraction. 
SECTION: Activation
10.4. Resuspend in 100 µl cold PBS. Alternatively, add proteinase K and RNase A directly to the activation reaction before column extraction. 
SECTION: Activation
10.5. Quality control: 
Check nuclei by Trypan blue staining to determine recovery and check integrity of nuclei if desired.

SECTION: DNA Extraction for N50 of ~17kb
11. Extract DNA from nuclei.

This protocol will result in N50 ~17 kb. If longer fragments are desired (N50 ~50-70 kb), refer to step 13 for the DNA extraction protocol that better preserves longer fragments. 
SECTION: DNA Extraction for N50 of ~17kb
11.1. Use the Monarch Genomic DNA Purification Kit. Follow protocol for genomic DNA isolation using cell lysis buffer. Include RNase A. NB. If fixation was performed, be sure to do the 56°C incubation for lysis for 1 hour (not just 5 minutes) to reverse crosslinks. 
SECTION: DNA Extraction for N50 of ~17kb
11.2. Perform two elutions: 100 µl and then 35µl. 

SECTION: DNA Extraction for N50 of ~17kb
11.3. Quantify DNA yield by Qubit dsDNA BR Assay Kit. 
SECTION: DNA Extraction for N50 of ~17kb
11.4. Concentrate by speedvac if necessary for 3 µg DNA in 48 µl for input to library prep.

SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12. Perform library preparation on DNA from previous steps and start sequencing run. 

This protocol will result in N50 ~17 kb. If longer fragments are desired (N50 ~50-70 kb), refer to step 14 for library preparation protocol that better preserves longer fragments. If you are not multiplexing samples, then use LSK-110. LSK-109 is only used to barcode for multiple samples on a flowcell. 

Follow Nanopore protocol for Native Barcoding Ligation Kit 1-12 and Native Barcoding Ligation Kit 13-24 with the following modifications:

SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.1. Load ~3 µg DNA into end repair.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.2. Incubate for 10 minutes at 20°C for end repair instead of 5 minutes.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.3. Load ~ 1 µg of end repaired DNA into barcode ligation.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.4. Double the ligation incubation time to at least 20 minutes.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.5. Elute in 18 µl instead of 26 µl following barcode ligation reaction cleanup to allow for more material to be loaded into the final ligation.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.6. Load ~3 µg of pooled barcoded material into the final ligation. If needed, concentrate using speedvac to be able to load 3 µg into the final ligation.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.7. Double the ligation incubation time to at least 20 minutes. 
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.8. Make sure to use LFB (NOT ethanol) for the final cleanup.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.9. Perform final elution in 13 µl EB. Take out 1 µl to dilute 1:5 for quantification by Qubit (and size distribution analysis by TapeStation / Bioanalyzer if desired).
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.10. Load ~1 µg of DNA onto the sequencer. 
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.11. Bubbles will absolutely destroy pores and ruin runs; mix and spin down all flush/wash solutions really well to eliminate bubbles.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.12. The Flow Cell Wash Kit can increase the throughput per flowcell with <1% carryover of pre-wash barcodes.
SECTION: Library Preparation and Sequencing Run for N50 of ~17kb
12.13. Spiking in more library + SQB + LB during a run, without a wash step, can also increase pore occupancy if it’s low. 
SECTION: DNA Extraction for N50 of ~50-70kb
13. Extract DNA from nuclei and preserve long fragments with N50 of ~50-70kb. 
SECTION: DNA Extraction for N50 of ~50-70kb
13.1. Use the NEB Monarch HMW DNA Extraction Kit. Follow protocol for genomic DNA isolation using cell lysis buffer. Include RNase A. Perform lysis with 2000 rpm agitation. We have validated 2000 rpm gives N50 ~50-70 kb but if longer reads are desired we expect 300 rpm would work.

Apart from using a different kit, all of the steps for the long fragment DNA extraction are the same as the general protocol. To reiterate, make the following changes to the protocol:
SECTION: DNA Extraction for N50 of ~50-70kb
13.2. If fixation was performed, be sure to do the 56°C incubation for lysis for 1 hour (not just 10 minutes) to reverse crosslinks. Agitate for 10 minutes and then keep at 56ºC without agitation for 50 minutes. 
SECTION: DNA Extraction for N50 of ~50-70kb
13.3. Quantify DNA yield by Qubit dsDNA BR Assay Kit. 
SECTION: DNA Extraction for N50 of ~50-70kb
13.4. Concentrate by speedvac if necessary for 3 µg DNA in 48 µl for input to library prep.

SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14. Perform library preparation on DNA from previous steps and start sequencing run.

This is a specialized library preparation and sequencing run that preserves long fragments and results in N50 ~50-70 kb. This protocol was adapted from “DNA extraction and Nanopore library prep from 15-30 whole flies” (Kim et al, dx.doi.org/10.17504/protocols.io.bdfqi3mw)

Follow Nanopore protocol for Ligation Sequencing Kit with the following modifications:

SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.1. Increase end preparation time to 1 hour with a 30 minute deactivation.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.2. Following end preparation, perform a cleanup by combining 60 μL SRE buffer from Circulomics (SS-100-101-01) with the 60 μL end prep reaction. 
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.3. Centrifuge this reaction at 10,000 x g at room temperature for 30 minutes (or until DNA has pelleted). 
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.4. Wash pelleted DNA with 150 μL of 70% ethanol two times, using a 2 minute spin at 10,000 x g between washes.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.5. Resuspend the pellet in 31 μL EB.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.6. Incubate at 50ºC for 1 hour. Incubate at 4ºC for at least 48 hours.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.7. For the ligation step, reduce ligation volume by half (total of 30 μL DNA in a 50 μL reaction volume). Increase the ligation incubation to 1 hour. 
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.8. Pellet DNA at 10,000 x g at room temperature for 30 minutes.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.9. Wash the pellet twice with 100 μL LFB, using a 2 minute spin at 10,000 x g between washes. 
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.10. Resuspend the pellet in 31 μL EB.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.11. Incubate at least 48 hours at 4ºC. 
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.12. Load 500 ng of DNA onto the sequencer.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.13. Bubbles will absolutely destroy pores and ruin runs; mix and spin down all flush/wash solutions really well to eliminate bubbles.
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.14. Use the Flow Cell Wash Kit (it can increase the throughput per flowcell with <1% carryover of pre-wash barcodes).
SECTION: Library Preparation and Sequencing Run for N50 of ~50-70kb
14.15. Reload the sequencer every 24 hours.