DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

Summary

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

Abstract

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

Introduction

DNA adenine methyltransferase identification (DamID) ^1,2 is a method to detect protein-DNA interactions in vivo and is an alternative approach to chromatin immunoprecipitation (ChIP) ³. It uses a relatively low amount of cells and does not require chemical cross-linking of protein with DNA or a highly specific antibody. The latter is particularly helpful when the target protein is loosely or indirectly associated with DNA. DamID has been successfully used to map the binding sites of a variety of proteins including nuclear envelope proteins ^4-10, chromatin associated proteins ^11-13, chromatin modifying enzymes ¹⁴, transcription factors and co-factors^15-18 and RNAi machineries ¹⁹. The method is applicable in multiple organisms including S. cerevisiae ¹³, S. pombe ⁷, C. elegans ^9,17, D. melanogaster ^5,11,18,20, A. thaliana ^21,22 as well as mouse and human cell lines ^6,8,10,23,24.

The development of the DamID assay was based on the specific detection of adenine-methylated DNA fragments in eukaryotic cells that lack endogenous adenine methylation ². An expressed fusion protein, consisting of the DNA-binding protein of interest and E. coli DNA adenine methyltransferase (Dam), can methylate the adenine base in GATC sequences that are in spatial proximity (most significantly within 1 kb and up to roughly 5 kb) to the binding sites of the protein in the genome ². The modified DNA fragments can be specifically amplified and hybridized to microarrays to detect the genomic binding sites of the protein of interest ^1,25,26. This original DamID method was limited by the availability of microarrays and the density of predetermined probes. We have therefore integrated high throughput sequencing into DamID ¹⁰ and designated the method as DamID-seq. The large number of short reads generated from DamID-seq enables precise localization of protein-DNA interactions genome-wide. We found that DamID-seq provided a higher resolution and a wider dynamic range than DamID by microarray for studying genome-nuclear lamina (NL) associations ¹⁰. This improved method allows probing NL associations within gene structures ¹⁰ and facilitates comparisons with other high throughput sequencing data, such as ChIP-seq and RNA-seq.

The DamID-seq protocol described here was initially developed for mapping genome-NL associations ¹⁰. We generated a fusion protein by tethering mouse or human Lamin B1 to E. coli DNA adenine methyltransferase and tested the protocol in 3T3 mouse embryonic fibroblasts, C2C12 mouse myoblasts ¹⁰ and IMR90 human fetal lung fibroblasts (data not published). In this protocol, we start with constructing vectors and expressing Dam-tethered fusion proteins by lentiviral infection in mammalian cells ²⁴. Next, we describe the detailed protocols of amplifying adenine-methylated DNA fragments and preparing sequencing libraries that should be applicable in other organisms.

Protocol

1. Generation and Expression of Fusion Proteins and Free Dam Proteins

1. Clone protein of interest into the DamID vector.
   1. Amplify cDNA of protein of interest (POI) using the desired high fidelity DNA polymerase and appropriate primers according to the manufacturer's protocol. Experimentally determine optimal amplification conditions to ensure proper amplification of inserts.
   2. Run an agarose gel and purify amplified cDNA of POI by the gel extraction kit according to the manufacturer's protocol.
   3. Clone cDNA of POI into the pDONR201 vector using BP Clonase II according to the manufacturer's protocol.
   4. Clone the cDNA of POI from the donor vector into the pLGW-RFC1-V5-EcoDam destination vector or the pLGW-EcoDam-V5-RFC1 destination vector ²⁷ using LR Clonase II according to the manufacturer's protocol, depending on the desired direction of fusing POI to the N-terminus or the C-terminus of the E. coli DNA adenine methyltransferase (EcoDam) ²⁷.
   5. Verify by sequencing that the cloned cDNA has a correct sequence and forms in-frame fusion to EcoDam.
2. Generate lentiviral stocks.
   1. Generate lentiviral stocks expressing Dam-V5-POI and V5-Dam (from the pLGW-V5-EcoDam vector ²⁷) using lentiviral expression systems. Use the forward transfection procedure according to the manufacturer's protocol.
      1. Use 293T cells and lipofection to generate lentiviral stocks according to the manufacturer's protocol for transfection.
      2. Include the 0.45 µm PVDF filter step.
3. Infect cells with lentivirus.
   1. The day before infection (Day 0), pass adherent cells cultured in appropriate growth media for this cell type to a 6-well tissue culture plate using the same growth media without antibiotics to achieve 50% confluence on the day of infection. Place cells in a 37 °C incubator.
   2. On the day of infection (Day 1), remove 2 cryovials of both Dam-V5-POI and V5-Dam lentiviral supernatants from a -80 °C freezer and place in a 37 °C water bath to thaw.
      1. Remove growth media from cells and replace with 0.5 ml of fresh growth media without antibiotics.
      2. Add 1 ml of the thawed lentivirus to each well (2 wells with V5-Dam and 2 wells with Dam-V5-POI). Add 1 ml of growth media without antibiotics to the remaining 2 wells-this will serve as a negative control. Gently shake the 6-well plate to mix and place back in a 37 °C incubator O/N.
   3. The day after infection (Day 2), remove viral suspensions from cells and replace with 2 ml growth media without antibiotics. Place cells back in a 37 °C incubator for 48 hr.
4. Isolate gDNA.
   1. Aspirate media from each well and detach cells using 250 µl 0.05% trypsin-EDTA. Incubate at 37 °C for 2 min.
   2. Wash cells off the plate with 1 ml growth media and pipette cells from each well into a 1.5 ml microcentrifuge tube. Pellet cells by centrifuging at 200 x g for 5 min at RT.
   3. Wash pelleted cells with 500 µl PBS and centrifuge at 200 x g for 2 min at RT.
   4. Resuspend pelleted cells in 200 µl PBS.
   5. Isolate gDNA by blood and tissue kit according to the manufacturer's protocol. Elute gDNA in 200 µl buffer AE and determine the concentration by measuring OD₂₆₀ using a spectrophotometer.
      Note: gDNA from uninfected or mock infected cells can be isolated as negative controls. Precipitate gDNA to higher concentrations for long-term storage.
      1. Add 3 volumes of 100% ethanol and 0.1 volume of 3 M sodium acetate (pH 5.5), and mix by inverting tubes 4-6 times.
      2. Store at -20 °C O/N.
      3. Centrifuge at 16,000 x g for 15 min at 4 °C.
      4. Carefully remove the supernatant. Wash pellets with 70% (volume/volume) ethanol and centrifuge at 16,000 x g for 5 min at 4 °C.
      5. Carefully remove ethanol and allow pellets to air dry for 3 min at RT.
      6. Dissolve gDNA in T₁₀E_0.1 (pH 7.5) to approximately 1 µg/µl. Pool gDNA from each experimental sample or the negative control and measure the concentration. Store at -20 °C.

2. Amplify Adenine-methylated DNA Fragments

1. Digest gDNA with DpnI that only cuts adenine-methylated GATCs.
   1. Set up a reaction on ice with 2.5 µg gDNA, 1 µl 10x buffer, 0.5 µl DpnI (20 U/µl) and fill with H₂O to a total volume of 10 µl. For each gDNA sample, prepare three reactions — one without DpnI ("no DpnI", replace DpnI with 0.5 µl H₂O) and two with DpnI ("with DpnI").
   2. Digest O/N at 37 °C and inactivate DpnI at 80 °C for 20 min.
2. Ligate DamID adaptors.
   1. Prepare DamID adaptors.
      1. Resuspend each of the two DamID adaptor oligos ²⁴ in H₂O to a final concentration of 100 µM.
      2. Combine equal volumes of the two DamID adaptor oligos, mix and place in a tightly closed tube. Seal the tube with Parafilm, sit in a rack and place in a beaker filled with water at 90 °C. Keep the water level below the cap of the tube (to avoid water getting into the tube) but above the surface of the oligo mix.
      3. Let the water cool to RT so the adaptors anneal slowly.
      4. Aliquot the annealed adaptors (50 µM) and store at -20 °C.
   2. Set up a reaction on ice. In each tube from 2.1.2, add 6.2 µl H₂O, 2 µl 10x ligation buffer, 0.8 µl 50 µM DamID adaptors (thawed on ice) and 1 µl T4 DNA ligase (5 U/µl). The total volume is 20 µl. In one of the two "with DpnI" tubes, replace ligase with 1 µl H₂O ("no ligase"). Note that each gDNA sample has two negative controls — " no DpnI " and " no ligase".
   3. Ligate O/N at 16 °C and inactivate ligase at 65 °C for 10 min.
3. Digest DNA with DpnII to destroy fragments that contain unmethylated GATCs.
   1. Set up a reaction on ice. In each tube from 2.2.3, add 24 µl H₂O, 5 µl 10x DpnII buffer and 1 µl DpnII (10 U/µl). The total volume is 50 µl.
   2. Digest at 37 °C for 2-3 hr and inactivate DpnII at 65 °C for 20 min.
4. Amplify adenine-methylated DNA fragments.
   1. Set up a reaction on ice with 5 µl DpnII digest from 2.3.2, 5 µl 10x PCR buffer, 12.5 µl 5 µM DamID PCR primer ²⁴, 4 µl 10 mM dNTP mix, 1 µl 50x polymerase mix and 22.5 µl H₂O. The total volume is 50 µl.
   2. Run the PCR as follows: 68 °C for 10 min; 94 °C for 1 min, 65 °C for 5 min, 68 °C for 15 min; 4 cycles of 94 °C for 1 min, 65 °C for 1 min, 68 °C for 10 min; 17 cycles of 94 °C for 1 min, 65 °C for 1 min, 68 °C for 2 min.
   3. Analyze 5 µl PCR products from each reaction on a 1% agarose gel. PCR products should appear as a smear ranging from 0.2 to 2 kb (Figure 2). The "no DpnI" and "no ligase" controls should have no or clearly less amplification.
   4. If the result from step 2.4.3 is satisfactory, repeat steps 2.4.1-2.4.3 with two or three reactions for the experimental sample and one reaction for each of the two negative controls.
   5. Pool and purify PCR products from the same experimental sample using PCR purification kits or Solid Phase Reversible Immobilization (SPRI) beads according to the manufacturer's protocol. Do not purify "no DpnI" or "no ligase" controls. Elute DNA with buffer EB.
   6. Measure the concentration of the purified DNA by measuring OD₂₆₀ using a spectrophotometer, which should be around 0.1 µg/µl or higher. Collect a minimum of 10 µg DNA for each sample. If using PCR purification kits, purify each 50 µl PCR products with one column, elute in 30 µl buffer EB and pool the eluates.
5. Digest DNA with DpnII to prevent DamID PCR primers from being sequenced.
   1. Set up a reaction on ice with 5 µg purified DNA from 2.4.6, 5 µl 10x DpnII buffer, 1 µl DpnII (10 U/µl) and fill with H₂O to a total volume of 50 µl. Prepare two or three reactions for each sample.
   2. Digest at 37 °C for 2-3 hr and inactivate DpnII at 65 °C for 20 min.
   3. Pool and purify the digests from the same sample with PCR purification kits or SPRI beads according to the manufacturer's protocol. Elute DNA with buffer EB.
   4. Measure the concentration of the purified DNA which should be around 0.06 µg/µl or higher. Collect a minimum of 6 µg DNA for each sample. If using PCR purification kits, purify each 50 µl digest with one column, elute in 30 µl buffer EB and pool the eluates.

3. Library Preparation for High-throughput Sequencing

1. Fragment DNA
   1. Experimentally determine the appropriate digestion time for each new batch of dsDNA Fragmentase. As the enzyme activity may decrease over time, repeat the test before performing a new experiment. To save DNA from 2.5.4 for the actual fragmentation, use purified methyl PCR products from 2.4.6 or extra DNA from previous experiments at this step.
      1. Set up a master mix with 6 µg DNA, 12 µl 10x Fragmentase buffer and fill with H₂O to a total volume of 114 µl.
      2. Vortex the Fragmentase stock vial for 3 sec, add 6 µl into the master mix and vortex the master mix for 3 sec. The total volume is 120 µl.
      3. Aliquot 20 µl of the master mix to each of 5 new tubes. Incubate all 6 tubes at 37 °C for 5 to 55 min at an increment of 10 min. Add 5 µl 0.5 M EDTA to stop the reaction.
      4. Analyze 12.5 µl digest (0.5 µg DNA) of each reaction as well as 0.5 µg undigested DNA on an agarose gel (Figure 3). Determine the minimal time (T_0.2kb) needed to digest the majority of the smear to around 0.2 kb. Select 6 time durations between 5 min and T_0.2kb (including 5 min and T_0.2kb) with equal increments for the actual fragmentation.
   2. Set up the actual fragmentation as described in 3.1.1.1-3.1.1.3. Incubate reactions at 37 °C for the time durations determined in 3.1.1.4.
   3. Pool 6 reactions and purify the digests with PCR purification kits or SPRI beads according to the manufacturer's protocol. Elute DNA in 51 µl buffer EB. The final eluate is ~50 µl.
2. Repair ends of the fragmented DNA
   1. Set up a reaction on ice with the eluate from 3.1.3, 25 µl H₂O, 10 µl 10x T4 DNA ligase buffer with 10 mM ATP, 4 µl 10 mM dNTP mix, 5 µl T4 DNA polymerase (3 U/µl), 1 µl Klenow DNA polymerase (5 U/µl), and 5 µl T4 polynucleotide kinase. The total volume is 100 µl. Mix well with pipette. Avoid foam and bubbles.
   2. Incubate at 20 °C for 30 min.
   3. Purify the DNA with PCR purification kits or SPRI beads according to the manufacturer's protocol. Elute DNA in 33 µl buffer EB. The final eluate is ~32 µl.
3. Add "A" overhangs
   1. Set up a reaction on ice with the eluate from 3.2.3, 5 µl 10x Klenow buffer, 10 µl 1 mM dNTP, and 3 µl Klenow (3'→5' exo-) (5 U/µl). The total volume is 50 µl. Mix well with pipette. Avoid foam and bubbles.
   2. Incubate at 37 °C for 30 min.
   3. Purify the DNA with PCR purification kits or SPRI beads according to the manufacturer's protocol. Elute DNA in 22 µl buffer EB. The final eluate is ~21 µl.
4. Ligate sequencing adaptors
   1. Prepare sequencing adaptors ²⁸.
      1. Resuspend each adaptor oligo to a concentration of 100 µM in 10 mM Tris-Cl (pH 7.8), 0.1 mM EDTA (pH 8.0) and 50 mM NaCl.
      2. Mix equal volumes of the universal adaptor ²⁸ and an indexed adaptor ²⁸.
      3. Anneal the adaptors in a thermal cycler with the following program: 95 °C for 2 min; 140 cycles for 30 sec starting at 95 °C and decreasing by 0.5 °C every cycle; hold at 4 °C.
      4. Aliquot adaptors and store at -20 °C.
   2. Set up a reaction on ice with the eluate from 3.3.3, 25 µl 2x ligation buffer, 1.5 µl 50 µM annealed sequencing adaptors (thawed on ice) from 3.4.1.4 and 2.5 µl T4 DNA ligase. Mix well with pipette. Avoid foam and bubbles. If multiplex sequencing is desired, use a different indexed adaptor for each sample.
   3. Incubate at RT for 1 hr.
   4. Purify the DNA with PCR purification kits or SPRI beads according to the manufacturer's protocol. Elute DNA in 24 µl buffer EB. The final eluate is ~23 µl.
5. Convert Y-shaped adaptors to dsDNA to enable accurately determining DNA fragment sizes ²⁸
   1. Set up a reaction on ice with the eluate from 3.4.4, 12.5 µl H₂O, 1 µl 25 µM primer 1 ²⁸, 1 µl 25 µM primer 2 ²⁸, 1.5 µl 10mM dNTP, 10 µl 5x PCR Buffer and 1 µl DNA polymerase (1 U/µl).
   2. Run the PCR as follows: 95 °C for 3 min; 5 cycles of 98 °C for 15 sec, 63 °C for 30 sec, 72 °C for 30 sec; 72 °C for 1 min; 4 °C on hold.
   3. Purify the DNA with PCR purification kits or SPRI beads according to the manufacturer's protocol. Elute DNA in 11 µl buffer EB. The final eluate is ~10 µl.
6. Size select the library
   1. Prepare a 2% agarose gel with 1x TAE buffer. Add ethidium bromide (CAUTION!) to a final concentration of 500 ng/ml when the melted TAE-agarose solution has cooled to avoid the inhalation of ethidium bromide. Make sure to have enough lanes for all the samples, DNA ladder and empty lanes.
   2. Add 8 µl 6x loading dye to the eluate from 3.5.3.
   3. Prepare DNA ladder by mixing 1 kb plus DNA ladder (1.0 µg/µl), 6x loading dye and H₂O in a ratio of 1:1:4.
   4. Load 6 µl DNA ladder, the samples from 3.6.2 and another 6 µl DNA ladder, each in a separate lane and with at least one empty lane from the adjacent samples/ladders.
   5. Run the gel at 120 V for 60 min.
   6. View the gel on a UV transilluminator (minimize the exposure time to UV). Wear safety glasses and a face shield. Make sure at least one of the DNA ladders run well with appropriate spacing (enough for excising 3 gel slices) between 300 bp and 400 bp bands. Narrow spacing increases the difficulties in excising multiple gel slices, while wide spacing increases the volume of excised gel slices.
   7. Use a new scalpel or a razor blade for each lane. Excise three thin gel slices between 300 and 400 bp from each lane (Figure 4) and place them each in a microcentrifuge tube. Keep the volume of each gel slice as low as possible (< 100 µl).
   8. Measure the volume of each gel slice (1 µl gel weighs approximately 1 mg), add 6x volumes of QG buffer and incubate at 50 °C.
   9. Vortex the QG-gel mixture every 2-3 min until the gel slice has completely dissolved. Add 2x gel volumes of isopropanol and mix.
   10. From this step, follow the protocol from gel extraction kits. Elute DNA in 51 µl buffer EB. The final eluate is ~50 µl.
7. Enrich sequencing adaptor modified DNA fragments
   1. Optimize the number of PCR cycles ²⁹.
      1. Set up a reaction on ice with 1 µl eluate from 3.6.10, 1 µl 25 µM primer 1 ²⁸, 1 µl 25 µM primer 2 ²⁸, 7 µl H₂O and 10 µl SYBR Green Supermix. The total volume is 20 µl.
      2. Run the qPCR as follows: 95 °C for 3 min; 20 cycles of 95 °C for 30 sec, 63 °C for 30 sec, 72 °C for 30 sec, plate read.
      3. Analyze data using a qPCR analysis software and determine the Quantification Cycle (Cq) or Threshold Cycle (Ct) for each sample using the manufacturer's protocol. Continue with the samples that have Cq/Ct ≤ 14. Use the maximal Cq (Cq₀) minus 1 (rounded up to the next higher integer) as the final PCR cycle number (N_PCR).
      4. Adjust the quantities of DNA templates so that different samples will be amplified to approximately equal amounts after running the same number of PCR cycles. Use 8 µl template for the sample with the highest Cq (Cq₀), and calculate the template volume of other samples with the following formula:
         Vol_i = 8 x 1.8^Cq_i-Cq₀
   2. Set up PCR reactions on ice with the template volumes calculated from 3.7.1.4: 1 µl 25 µM primer 1 ²⁸, 1 µl 25 µM primer 2 ²⁸, 1.5 µl 10 mM dNTP, 10 µl 5x buffer, 1 µl DNA polymerase (1 U/µl) and fill with H₂O to a total volume of 50 µl.
   3. Run the PCR as follows: 95 °C for 45 sec; N_PCR cycles (determined in 3.7.1.3) of 98 °C for 15 sec, 63 °C for 30 sec, 72 °C for 30 sec; 72 °C for 1 min; 4 °C on hold.
   4. Analyze 5 µl PCR products in a 2% agarose gel (Figure 5A). A clear "single" band indicates that the amplified DNA fragments are within a narrow length range and that the DNA library can be subject to further analysis.
   5. Repeat one reaction for selected samples and pool the amplified DNA libraries from the same sample.
   6. Purify selected DNA libraries for sequencing.
      1. If primer/adaptor dimers are not visible in 3.7.4, purify DNA libraries with PCR purification kits or SPRI beads according to the manufacturer's protocol. Elute DNA in 21 µl buffer EB. The final eluate is ~20 µl. If the user's sequencing facility has specific instructions on the elution buffer, the final volume, etc., prepare samples accordingly.
      2. If primer/adaptor dimers are clearly visible in 3.7.4, purify libraries as follows.
         1. Load DNA from 3.7.5 and DNA Ladder in a 2% precast agarose gel. Samples can be purified as described in 3.7.6.1 to reduce the volume or can be loaded into multiple wells. Place the agarose gel in an appropriate power system. Allow the gel to run for 15 min.
         2. Using an appropriate transilluminator, cut out the desired band with a clean scalpel/razor for each sample and place the gel slice in a 1.5 ml microcentrifuge tube.
         3. Isolate DNA using gel extraction kits according to the manufacturer's protocol with two modifications: incubate buffer QG-gel mixture in a thermo-mixer at 37 °C and 1,000 rpm for 30 min, and add 2x gel volumes of isopropanol.
8. Submit purified DNA libraries to a sequencing facility. Follow all facility guidelines.
   Note: A quality analysis by a Bioanalyzer (Figure 5B) should be performed prior to sequencing in order to determine the exact size range and the concentration of a DNA library.

Results

The Dam-V5-LmnB1 fusion protein was verified to be co-localized with the endogenous Lamin B protein by immunofluorescence staining (Figure 1).

The successful PCR amplification of adenine-methylated DNA fragments is a key step for DamID-seq. The experimental samples should amplify a smear of 0.2 - 2 kb while the negative controls (without DpnI, without ligase or without PCR template) should result in no-or clearl...

Discussion

Whether Dam-tagged proteins retain the functions of endogenous proteins should be examined before a DamID-seq experiment. The subcellular localization of Dam-tagged nuclear envelope proteins should always be determined and compared with that of the endogenous proteins. For studying transcription factors, it is suggested to examine whether the Dam-fusion protein can rescue the functions of the endogenous protein in regulating gene expression. This functional test can be performed in organisms in which knockout mutants of ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ViraPower Lentiviral Expression Systems	Life Technologies	K4950-00, K4960-00, K4970-00, K4975-00, K4980-00, K4985-00, K4990-00, K367-20, K370-20, and K371-20
Gateway BP Clonase II Enzyme Mix	Life Technologies	11789-020
Gateway LR Clonase II Enzyme Mix	Life Technologies	11791-020
DNeasy Blood & Tissue Kit (250)	QIAGEN	69506 or 69504
Gateway pDONR 201	Life Technologies	11798-014
293T cells	American Type Culture Collection	CRL-11268
Trypsin-EDTA (0.05%), phenol red	Life Technologies	25300-054
DMEM, high glucose, pyruvate	Life Technologies	11995-065
Fetal Bovine Serum	Sigma	F4135
Tris			brand not critical
EDTA			brand not critical
200 Proof EtOH			brand not critical
Isopropanol			brand not critical
Sodium Acetate			brand not critical
DpnI	New England Biolabs	R0176	supplied with buffer
DamID adaptors "AdRt" and "AdRb"	Integrated DNA Technologies		sequences available in ref. 24; no phosphorylation of the 5' or 3' end to prevent self-ligation.
T4 DNA Ligase	Roche Life Science	10481220001	supplied with buffer
DpnII	New England Biolabs	R0543	supplied with buffer
DamID PCR primer "AdR_PCR"	Integrated DNA Technologies		sequences available in ref. 24
Deoxynucleotide (dNTP) Solution Set	New England Biolabs	N0446	100 mM each of dATP, dCTP, dGTP and dTTP
Advantage 2  Polymerase Mix	Clontech	639201	supplied with buffer
1Kb Plus DNA Ladder	Life Technologies	10787018	1.0 µg/µl
QIAquick PCR Purification Kit	QIAGEN	28104 or 28106
MinElute PCR Purification Kit	QIAGEN	28004 or 28006	for an elution volume of less than 30 µl
SPRI beads / Agencourt AMPure XP	Beckman Coulter	A63880	apply extra mixing and more elution time if less than 40 µl elution buffer is used
Buffer EB	QIAGEN	19086
NEBNext dsDNA Fragmentase	New England Biolabs	M0348	supplied with buffer
T4 DNA Ligase Reaction Buffer	New England Biolabs	B0202
T4 DNA Polymerase	New England Biolabs	M0203
DNA Polymerase I, Large (Klenow) Fragment	New England Biolabs	M0210
T4 Polynuleotide Kinase	New England Biolabs	M0201
Klenow Fragment (3’ → 5’ exo-)	New England Biolabs	M0212	supplied with buffer
sequencing adaptors	Integrated DNA Technologies		sequences available in ref. 28
Quick Ligation Kit	New England Biolabs	M2200	used in 3.4.3; supplied with Quick Ligation Reaction Buffer and Quick T4 DNA Ligase
sequencing primer 1 and 2	Integrated DNA Technologies		sequences available in ref. 28
KAPA HiFi PCR Kit	Kapa Biosystems	KK2101 or KK2102	supplied with KAPA HiFi DNA Polymerase, 5x KAPA HiFi Fidelity Buffer and 10 mM dNTP mix
agarose	Sigma Aldrich	A4679
ethidium bromide	Sigma Aldrich	E1510-10ML	10 mg/ml
QIAquick Gel Extraction Kit	QIAGEN	28704 or 28706
iTaq Universal SYBR Green Supermix	Bio-Rad Laboratories	1725121 or 1725122
Spectrophotometer			brand not critical
0.45 μm PVDF Filter			brand not critical
25 ml Seringe			brand not critical
10 cm Tissue Culture Plates			brand not critical
6-well Tissue Culture Plates			brand not critical
S1000 Thermal Cycler	Bio-Rad Laboratories
C1000 Touch Thermal Cycler	Bio-Rad Laboratories		for qPCR
Vortex Mixer			brand not critical
Dry Block Heater or Thermomixer			brand not critical
Microcentrifuge			brand not critical
Gel electrophoresis system with power supply			brand not critical
Magnet stand			for purification of DNA with SPRI beads; should hold 1.5-2 ml tubes; brand not critical
UV transilluminator			brand not critical
E-gel electrophoresis system	Life Technologies	G6400, G6500, G6512ST