Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Resumo

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Resumo

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Introdução

In eukaryotic organisms, genome duplication is initiated from multiple sites called replication origins, where the origin recognition complex (ORC) binds and initiates the recruitment protein machinery, including the CMG helicase, to initiate DNA replication¹. The replisome machinery at each replication origin forms two replication forks that move bidirectionally^2,3. The twin replication forks replicate the two parent strands of DNA through the continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand. Given this asymmetric nature of each replication fork, the replication components, including the DNA polymerases on the leading and lagging strands, are different. Previous genetic analysis based on mutant DNA polymerases demonstrates that DNA Pol ε and DNA Pol δ are the major polymerases responsible for leading and lagging strand replication, respectively^4,5. Several methods have been developed to detect the DNA polymerase's strand preference, including Pu-seq^6,7, HydEn-seq⁸, and emRiboSeq or RiboSeq^9,10. These genetic methods to dissect the strand specificity are limited to DNA polymerases and require introducing mutations on each protein^4,5,11,12. Tracking the association of any target protein with leading and lagging strands of DNA replication forks will provide valuable insights into DNA replication. In this effort, we first developed the eSPAN technique (enrichment and sequencing of protein-associated nascent DNA, Figure 1) in yeast cells. As a proof-of-principal study, we first showed that Pol δ and Pol δ are enriched on leading and lagging strands, respectively. We also uncovered the strand preference of key DNA replication components, including the replicative DNA helicase, the single-strand DNA binding protein (replication protein A), and the proliferating cell nuclear antigen (PCNA) lamp and its loader replication factor C (RFC)¹³. Importantly, we and others have used this method to uncover mechanisms of parental histone transfer, a process that was intractable for over 4 decades^14,15,16,17.

The eSPAN method involves two sequential steps: enrichment of the target protein by ChIP¹⁸ or CUT&Tag¹⁹ and enrichment of the nascent DNA associated with the protein by BrdU immunoprecipitation. In a standard yeast strain (W303 or S288C), extracellular BrdU or EdU cannot enter cells and incorporate into chromosomes. These strains lack an appropriate nucleoside transporter for thymidine uptake as well as the thymidine kinase required to phosphorylate thymidine into TMP, which converts BrdU (or EdU) to BrdUMP (EdUMP). Expression of human equilibrative nucleoside transporter 1 (hENT1) and herpes simplex virus thymidine kinase (HSV-TK) enhances thymidine uptake and the conversion of BrdU (or EdU) to BrdUMP (EdUMP)²⁰. Therefore, to perform the eSPAN experiment in yeast, the strains had to be inserted with a BrdU-Incorporating (BrdU-Inc) structure (HSV-TK and hENT1)^13,21.

Previously, we provided the eSPAN protocol for DNA replication proteins in yeast²². Recently, we integrated the CUT&Tag method into the eSPAN protocol in mammalian cells to address technically challenging steps that require optimization, such as cross-linking, chromatin shearing, and to minimize the number of starting cells^17,23. Here, we provided histone eSPAN for visualization using yeast cells (Figure 2). This method is primarily used to analyze the distribution of modified forms of histones on replicating DNA strands. While we tested several histone marks, we used H3K4me3, a mark on the parental histone H3, as an example. To analyze replisome components and factors involved in DNA repair at replication forks, we recommend shearing chromatin into small fragments using sonication as described in our first publication²². For mammalian cells, we adopted the modified Cut&Tag procedures to replace the ChIP step described in yeast²³. This modification reduces the amount of starting material (cells) and, most importantly, enables the generation of eSPAN libraries for sequencing without the use of single-stranded DNA preparation kits. For the yeast protocol, we opted not to use the Cut&Tag method because the amount of starting material was sufficient. More importantly, we found that the Cut&Tag procedures were not consistently effective for yeast cells in our hands.

Protocolo

NOTE: For buffer and reagent preparation guidelines, see buffer and reagent preparation guidelines (Supplementary File 1).

1. Histone eSPAN in budding yeast

NOTE: Most yeast strains lack a functional nucleoside transporter for thymidine uptake. To efficiently label DNA with BrdU or EdU for eSPAN, a thymidine kinase-positive (TK+) strain²¹ is required. Additionally, only mating type 'A' yeast strains can be used for cell cycle arrest using alpha factor.

1. Yeast cell synchronization and BrdU labeling (8 h)
   1. Inoculate a single colony of appropriate yeast strain (TK+) into 10 mL of YPD media and grow at 30 °C on a shaking incubator overnight at 220 rpm.
   2. Measure the optical density, OD600 nm, with a spectrophotometer and dilute the culture into fresh 200 mL of YPD medium with OD600 ~0.2-0.3.
   3. Grow the culture up to OD600 ~0.4-0.5 (usually takes 3-4 h) at 30 °C in a shaking incubator at 220 rpm.
   4. Add 300 µL of α- factor (5 mg/mL) and culture for 1.5 h at 25 °C, 220 rpm.
   5. Add another dose of 300 µL of α-factor (5 mg/mL) and grow for another 1.5 h at 25 °C, 220 rpm.
   6. Prepare the 37.5% paraformaldehyde fixation solution and prewarm the medium (200 mL YPD +HU (hydroxyurea)+ BrdU) to 30°C during the cell synchronization.
      CAUTION: Handle BrdU with caution as it is a potential mutagen and teratogen. Prepare a 37.5% paraformaldehyde solution in a chemical fume hood to avoid exposure to harmful fumes, as paraformaldehyde is a potential carcinogen.
   7. Check the morphology of the cells (a bar shape) with a light microscope to ensure that at least 90% of the yeast cells are in the G1 phase 15 min before the 3 h G1 arrest. Alternatively, perform fluorescence-activated cell sorting (FACS) analysis to confirm G1 arrest²⁴.
      NOTE: Check cell morphology and confirm G1 arrest (90% with a bar shape) in this step.
   8. Collect the cells (pellet) by centrifugation at 1600 g for 5 min at 4 °C and wash the cell pellet twice with 20 mL of ice-cold double distilled water.
   9. Resuspend the cells in prewarmed (30 °C) 200 mL of YPD +HU+ BrdU.
      NOTE: The purpose of HU release is to ensure that most yeast cells are in the early replication S phase, which will improve consistency and ensure reproducibility of results. Omitting HU is acceptable if working with log phase cells is required.
   10. Culture the cells on a shaking incubator at 30 °C, 220 rpm for 45 min.
       NOTE: FACS analysis can be performed to confirm the early S phase. For FACS analysis, it is important to include a log-phase sample as a control to accurately define the G1 cell population. For efficient BrdU incorporation, it is advantageous to start with fast-growing cells. Some mutants with slow growth defects tend to show poor BrdU labeling efficiency. The efficiency of BrdU incorporation and performance of BrdU-IP is assessed by quantitative PCR by comparing replicated and unreplicated regions²² using DNA isolated from step 1.6.7.
2. Sample fixation and collection (1 h)
   1. Add freshly prepared 6 mL of 37% paraformaldehyde to a final concentration of 1%, mix well, and incubate at 25 °C for 20 min with gentle shaking, 90 rpm.
   2. Quench the fixation by adding 12 mL of 2.5 M glycine, mix well, and incubate at 25 °C for 5 min with gentle shaking, 90 rpm.
   3. Pellet the cells at 1600 g for 5 min at room temperature (RT). Then, wash the cells twice with 20 mL of cold water by spinning at 1600 g for 5 min.
   4. Collect the cells in 2 mL screw cap tubes (50 mL of cells/tube) and store them at -80 °C.
      ​NOTE: Cells can be stored for up to a year (pause point).
3. Yeast cell lysis and chromatin fragmentation (5 h)
   1. Thaw the cell pellets, resuspend by gentle vortexing, and wash with 0.4 mL of ChIP lysis buffer with protease inhibitors and antibiotic mix (preventing the growth of contaminating bacteria) and spin at 5 000 g for 1 min.
   2. Add 0.1 mL of ChIP lysis buffer containing protease inhibitors and an antibiotic mix, followed by approximately 100 µL of glass beads (0.5 mm).
   3. Lyse by bead beating for 3 cycles (30 s on/off, 1 min off) in a 4 °C cold room.
   4. Punch holes in the bottom of the tube with a 16 G hot needle.
      NOTE: To keep the hot needle, try to keep the tube close to fire.
   5. Collect the lysate by nesting the tube into a 1.5 mL empty DNA low binding tube and centrifuge at 1600 g for 2 min, RT.
   6. Aspirate the supernatant carefully without disturbing the pellet (chromatin will be in the cell debris fraction).
      CAUTION: Be careful not to accidentally aspirate the cell pellet when using the vacuum.
   7. Resuspend the cell pellets by pipetting in 0.35 mL of MNase Digestion Buffer supplemented with 0.5 mM spermidine and 0.1 mM 2-mercaptoethanol.
   8. Add 10 units of MNase to the reactions, mix them gently by inverting 4-6 times immediately, and incubate at 37 °C water bath for 20 min.
      NOTE: Digestion conditions should be optimized for each batch of MNase enzyme to enrich for mono-, di- or tri-nucleosome fractions.
   9. Quench the MNase digestion by adding 5 µL of 0.5 M EDTA for a final concentration of 10 mM EDTA. Mix them gently by inversion and keep tubes immediately in ice for at least 30 min.
      NOTE: Samples can be stored at 4 °C overnight at this step. MNase digestion conditions should be optimized to obtain the majority of mono-, di- or tri-nucleosome fractions (140-450 bp)
   10. Sonicate the reaction mixture with bioruptor microtubes (1.5 mL) for 3 min (high power, 30 s on/off). Spin at 10,000 g for 5 min at 4 °C.
       NOTE: This brief sonication helps release the chromatin into the buffer.
   11. Transfer the supernatant to new tubes and spin another 10 min at 10,000 g at 4 °C.
   12. Collect supernatant (about 400 µL), save 30 µL of the sample as input DNA, and freeze at -20 °C. Use the remaining (~370 µL ) cell lysate in the next chromatin immunoprecipitation (step 1.4.1).
4. Histone chromatin immunoprecipitation (overnight)
   1. Add 0.5 µg of H3K4me3 antibody to the lysate and nutate on the rotor, 10 rpm at 4 °C overnight.
      NOTE: Here, H3K4me3 is used as an example. The antibody amount should be experimentally tested.
   2. Spin at 15,800 ×  g for 10 min at 4 °C and transfer the supernatant to a new 1.5 mL tube.
   3. Add 20 µL of prewashed protein G Sepharose beads (3 times washed with ChIP lysis buffer, nutate on the rotor for 3 min each, collect beads by centrifugation at 350 × g, 4 °C) and incubate at 4 °C for 2 h.
   4. Spin at 350 × g at RT, and wash beads according to the following order.
      1. Wash twice with 1 mL of ice-cold lysis buffer for 3 min each time by rotation at 10 rpm on a rotator at RT.
      2. Wash once with 1 mL of ice-cold lysis buffer with 0.5 M NaCl buffer for 3 min by rotation at 10 rpm on a rotator at RT.
      3. Wash once with 1 mL of ice-cold Tris/LiCl washing buffer for 3 min by rotation at 10 rpm on a rotator at RT.
      4. Wash one with 1 mL of RT Tris/EDTA washing buffer (not TE buffer, Supplementary File 1 for the recipe). Sock all the possible liquid out from the pellet with a 16 G 1/2 needle.
5. DNA extraction (2 h)
   1. Add 0.1 mL of well-mixed 10% chelating resin for DNA extraction to the washed beads (from 1.4.4.4. step), input sample (from step 1.3.12), and vortex.
   2. Boil for 10 min on a 100 °C heat block, then cool to RT to reverse-crosslink.
      NOTE: Stop-pop locking clips for centrifuge tubes are strongly recommended to maintain the closure seal.
   3. Add 5 µL of 20 mg/mL protease K to the beads and incubate at 55 °C for 30 min while shaking at 400 rpm.
   4. Boil the beads on a 100 °C heat block for another 10 min to inactivate protease K activity.
      NOTE: Stop-pop locking clips for centrifuge tubes are strongly recommended to maintain the closure seal.
   5. Spin down the beads at 6000 g for 2 min at RT, and save the supernatant.
   6. Add 100 µL of 2XTE to the pellet. Vortex and spin down at 6000 g for 2 min at RT.
   7. Mix and save the supernatants (from steps 1.5.5 and 1.5.6) taken above (about 180 µL).
   8. Purify 30 µL samples into 18 µL of elution buffer (EB, supplied with PCR purification kit) using DNA purification columns for ssDNA library preparation (from step 1.3.12 as input and step 1.5.7 as H3K4me3 ChIP-seq samples).
      NOTE: DNA samples can be stored at -20 °C for up to a month (pause point).
6. BrdU immunoprecipitation (5 h)
   1. Boil samples (about 150 µL input and H3K4me3 ChIP from step 1.5.7) for 5 min on a 100 °C heat block, and immediately chill in ice for 5 min. Add 15 µL of 10x PBS and + 1.35 mL of BrdU IP buffer + 0.5 µL of anti-BrdU Ab + 1 µL of E.coli tRNA (10 mg/mL).
      NOTE: This is a critical step. Use the low DNA binding centrifuge tubes to avoid nonspecific DNA binding.
   2. Nutate for 2 h on a rotor at 4 °C at 10 rpm.
   3. Transfer the mixture to a 1.5 mL tube containing 20 µL of protein G Sepharose beads (washed three times with 1 mL of BrdU IP buffer and rotate for 3 min each at 10 rpm for each wash at RT) and nutate for 1 h at 4 °C on the rotor at 10 rpm.
   4. Spin at 800 × g for 1 min at RT and wash according to the following: three times with 1 mL of cold BrdU IP buffer, 3 min on a rotor, and once with 1 mL of TE buffer for 3 min.
   5. Spin at 800 × g for 1 min at RT and aspirate the supernatant.
   6. Add 100 × µL of TE + 1% SDS buffer into the remaining beads. Incubate at 65 °C for 5 min. Spin at 800 × g for 1 min at RT.
   7. Purify the supernatant into 18 µL of EB using DNA purification columns (BrdU IP and H3K4me3 eSPAN sample).
      NOTE: Use the low-binding centrifuge tubes to avoid nonspecific DNA binding. DNA samples can be stored at -20 °C for up to a month (pause point).
7. ssDNA library preparation and sequencing (3 h)
   1. Prepare the single-stranded DNA libraries with 15 µL of purified input, BrdU IP, H3K4me3 ChIP and eSPAN samples (steps 1.5.8 and 1.6.7) (including the adaptase, extension and ligation steps) according to manuals provided by ssDNA and low-input DNA library prep kit and purify the obtained ligation product into 20 µL of low-EDTA elution buffer (provided by DNA Library Prep Kit).
      NOTE: There are two ligation purification steps with the kit. This protocol follows the 200 bp, <1 ng procedure.
   2. Amplify DNA library using a total 50 µL reaction mix (reagents provided by ssDNA library prep kit): 20 µL of purified DNA library, 4 µL of reagent W2, 10 µL of reagent W3, 1 µL of enzyme W4, 1.5 µL of Indexes I7 primers (10 µM, see Supplemental Oligo Sequence) and 1.5 µL of indexes I5 primer (10 µM, see Supplemental Oligo Sequence), 16 µL of DNase-free ddH₂O. Heat the PCR mix at 98 °C for 30 s, followed by 18 cycles of 10 s at 98 °C, 30 s at 60 °C, and 1 min at 68 °C, and one cycle of 10 min at 68 °C.
      NOTE: Each library should utilize a unique combination of i7 and i5 primers. The number of amplification cycles should be adjusted based on the quantity of the library to avoid overamplification, which can result in PCR artifacts. Given the typically low DNA quantity in eSPAN samples, use up to 18 cycles.
   3. Purify the PCR products by mixing in 60 µL (1.0x volume) of beads (prewarmed to 30 °C) (see Table of Materials).
   4. Mix the PCR products and beads thoroughly.
      1. Let it stand at RT for 5 min. Bind beads on the magnetic stand for 3 min and wash the beads twice with (each time 200 µL) freshly prepared 80% ethanol.
      2. Air dry the beads for 2 min and elute into 20 µL low-EDTA buffer (provided in DNA Library Prep Kit).
   5. Measure the library concentration and fragment size distribution using high-resolution electrophoresis systems (see Table of Materials).
   6. Pool an equal amount of individual libraries, including the input, H3K4me3 ChIP, BrdU IP, and eSPAN samples, and perform parallel paired-end sequencing with the Illumina sequencing platform. For the data analysis process, refer to section 3.
      NOTE: The evaluation criteria for quality control results are: (i) No adapter/primer-dimer peaks (below 180 bp) are detected. (ii) All libraries to be pooled have a similar and narrow size distribution (~300 bp width). Overdrying the beads may result in low yield; use a DNA workstation to avoid contamination. Libraries can be stored indefinitely at -20˚C (pause point).

2. Histone eSPAN with mouse ES cell

NOTE: The histone eSPAN with mouse ES cell is essentially identical to a previously published protocol²³.

1. BrdU labeling and sample collection for mouse ES cells (3 h)
   1. Grow mouse ES cells in an ES cell culture medium at 37 °C with a humidified, 5% CO₂ atmosphere to reach 70%-80% confluency. In general, about 1 million cells were required for histone eSPAN. Seed cells 1 day before the experiments to ensure cells are growing at an exponential stage.
   2. Change the medium and incubate cells with BrdU at a final concentration of 50 µM for 30 min at 37 °C with a humidified, 5% CO₂ atmosphere.
      NOTE: BrdU labeling efficiency can be monitored through quantitative PCR analysis. Alternatively, immunofluorescence or FACS analysis can be used to monitor BrdU incorporation efficiency. Unlabeled cells can be used as a negative control. These are the standard labeling conditions for mouse ES cells. Adjusting the labeling conditions accordingly for cells with varying proliferation rates and S-phase duration is recommended.
   3. Rinse the cells twice gently with ice-cold PBS.
   4. Trypsinize and collect the cells by pelleting at 200 × g for 5 min.
   5. Gently pipette the cells to medium to resuspend them, then count them. Centrifuge the necessary number of cells at 500 × g for 5 min at RT.
   6. Resuspend the cell pellet in 4 mL of wash buffer by pipetting and discard the supernatant. Centrifuge at 500 × g for 5 min at RT.
   7. Resuspend the pellets again with 4 mL of wash buffer and centrifuge at 500 × g for 5 min at RT.
   8. Discard the supernatant and resuspend the cells in wash buffer.
   9. Place the ConA-coated magnetic beads (use 10 µL of beads for 250000 cells; scale up when using more cells) and resuspend in 1 mL of binding buffer. After multiple inversions, set the tubes on the magnetic stand to allow the beads to settle.
   10. Using a pipette or low-speed vacuum, discard the supernatants. Repeat the wash steps twice and resuspend the beads in the binding buffer.
   11. Add pre-washed ConA-coated magnet beads to the samples. Mix well and rotate at RT for 10-20 min.
   12. Place the tubes on a magnet stand to discard all the supernatants.
2. Tagmentation¹⁹ for mouse ES cells (2 days)
   1. Resuspend the beads (ConA-coated magnet beads bound with cells from step 2.1.10) in 200 µL of ice-cold antibody buffer. Add IgG as a negative control or the appropriate concentrations of primary antibodies against the proteins of interest.
   2. Mix well and nutate at 4 °C overnight.
   3. Mix 2 µg of the secondary antibody and a proper amount of pA-Tn5 at 1:2 molar ratio. Bring the total volume to 8 µL/sample with 50% (v/v) glycerol. Pipette several times to mix well and incubate at RT for 1 h.
   4. Place the CUT&Tag samples on a magnet stand for 30 s and discard all the supernatants. Add 1 mL of Dig-wash buffer to the beads pellet and invert several times.
      NOTE: From this to the following washing steps (steps 2.2.5-2.2.6), incubating the beads in the buffers for 3-5 min after the inverting step to avoid nonspecific binding is recommended.
   5. Place the tubes on a magnet stand and remove all the supernatants. Add 200 µL of Dig-300 buffer to resuspend the beads and add the 8 µL of the mixture of secondary antibody and pA-Tn5 to each sample. After mixing well with pipetting several times, incubate at RT for 1 h.
   6. Place the tubes on a magnet stand and remove all the supernatants. Add 1 mL of Dig-300 buffer to resuspend the beads and invert several times.
   7. Place the tubes on a magnet stand and remove all the supernatants. Repeat the washing step for 3 more times.
   8. Remove the supernatants completely and add 250 µL of Tagmentation buffer to resuspend the beads and incubate at 37 °C for 1 h.
      NOTE: Gentle shaking is recommended for the reaction step on a thermomixer because the beads may sediment to the bottom of the tubes.
   9. Place the tubes on a magnet stand and remove all the supernatants. Use 1 mL of TAPS wash buffer to resuspend the beads and invert several times.
   10. Place the tubes on a magnet stand and discard all the supernatants.
   11. Add 250 µL of Dig-300 buffer containing 15 mM EDTA, 0.1% (w/v) SDS, and 0.1 mg/mL proteinase K into the remaining beads. Vortex to mix well and incubate at 37 °C overnight with gentle shaking.
3. Genomic DNA purification and in vitro tagmentation in mouse ES cells (overnight)
   NOTE: Genomic DNA from the same cells for eSPAN is extracted and set up for in vitro tagmentation, which is used for BrdU-IP-ssSeq. The genomic DNA tagmentation and its following BrdU-IP-ssSeq are for eSPAN normalization purposes.
   1. Resuspend the cells in 300 µL of genomic DNA lysis buffer and vortex at maximum speed until no pellets are visible.
   2. For every sample, add RNase A at 100 µg/mL. Once combined, incubate at 37 °C for 10 min.
   3. Add 100 µL of protein precipitation solution to each sample. Mix well by vortexing and centrifuge at 15,000 g at RT for 3 min.
   4. Save the supernatant by transferring it to new 1.5-mL tubes. Add 1x volume isopropanol to the samples and mix well.
   5. Spin at maximum speed at RT for 3 min to pellet the genomic DNA. Carefully pour off the supernatants.
   6. Add 1 mL of freshly prepared 70% (v/v) ethanol to each sample and invert several times. Centrifuge at maximum speed at RT for 3 min.
   7. Centrifuge at 15,000 g for 1 min at RT and remove the remaining ethanol using a P20 pipette. Allow to air dry for 5-10 min.
   8. Add 50 µL of ddH₂O and vortex briefly to resuspend the genomic DNA. Dissolve at 65 °C for 30 min.
   9. Centrifuge briefly and transfer the supernatants to new 1.5 mL tubes. Quantify DNA concentration by spectrophotometer.
   10. Set up the in vitro tagmentation reaction as listed in Table 1.
   11. Mix well and incubate for 30 min at 37 °C with gentle shaking.
   12. Add 10 mM EDTA, 0.1% (w/v) SDS, and 0.1 mg/mL Proteinase K to each sample. Mix well by vortexing and incubate overnight at 37 °C with shaking.
4. DNA extraction and oligo-replacement (3 h)
   ​NOTE: Both phenol-chloroform-isoamyl alcohol (PCI) and column purification can be applied for DNA extraction. Here, detailed procedures using column purification are provided.
   1. Add 1.25 mL (5x volume) of binding buffer (supplied with PCR purification kit) to the samples (steps 2.2.11. and 2.3.12.) and mix well by inverting several times.
   2. Load the mixture to DNA purification columns (in 2-mL collection tubes) and centrifuge for 30 s at 10,000 × g at RT. Discard the flow-through.
   3. Add 600 µL elution buffer (included in the kit) to columns and centrifuge for 1 min at 10,000 × g at RT. Discard the flow-through.
   4. Centrifuge at maximum speed for 2 min to completely remove the remaining buffer. Place the columns in new 1.5-mL tubes with open lids and air dry for 1 min.
   5. Add 32 µL of DNase-free ddH₂O (or 10 mM Tris·HCl, pH = 8.0) to the middle of the membrane and allow it to rest for 1 min. Centrifuge at maximum speed for 2 min and the CUT&Tag DNA is now in solution.
   6. Add 32 µL of CUT&Tag DNA sample to a new PCR tube and then add 2 µL of dNTP mix (2.5 mM each), 4 µL of 10x Ampligase buffer, and 2 µL of rep-B (10 µM). Tap gently and mix by pipetting.
   7. Briefly spin down and run the following program for oligo replacement²³.
      50 °C, 1 min
      45 °C, 10 min
      Ramp to 37 °C, 0.1 °C/s
      ​37 °C, hold
   8. In the thermocycler, add 1 µL of T4 DNA polymerase and 1 µL of Ampligase to each tube (final volume, 40 µL/sample). Mix by gentle pipetting.
   9. Continue the reaction at 37 °C in the thermocycler for 30-60 min.
      NOTE: DNA can be stored at -20 °C if not proceeding to BrdU IP right after this step (safe pause point).
5. BrdU immunoprecipitation (essentially the same as the yeast protocol) (2 h)
   1. Transfer the samples to 1.5 mL DNA low-binding tubes. Boil samples 5 min on a 100 °C heat block, and immediately chill in ice for 5 min. Add 15 µL of 10x PBS and + 1.35 mL of BrdU IP buffer + 0.5 µL of anti-BrdU Ab + 1 µL of E.coli tRNA (10 mg/mL).
   2. Nutate for 2 h on a rotor at 4 °C.
   3. Transfer the mixture to a tube containing 20 µL of protein G Sepharose beads (washed three times with 1 mL of BrdU IP buffer, each 3 min).
   4. Incubate at 4 °C for 1 h.
   5. Spin at 800 × g for 1 min and wash according to the following: three times with 1 mL of cold BrdU IP buffer, 3 min on a rotor; once with 1 mL of TE buffer for 3 min.
   6. Spin at 800 × g for 1 min and aspirate the supernatant.
   7. Add 100 µL of TE + 1% SDS buffer into the remaining beads. Incubate at 65 °C for 15 min. Spin at 800 × g for 1 min.
   8. Purify the supernatant into 18 µL of elution buffer (supplied with kit) using DNA purification columns (BrdU IP and H3K4me3 eSPAN sample).
6. PCR amplification and sequencing (1.5 h)
   1. Amplify and label single-stranded-DNA library samples using different index combinations. Prepare the PCR reaction by adding the following components ((Purified DNA [ChIP or ESPAN], I5-Indexing primer [1 µL], I7-Indexing primer [1 µL], NEBNext High-Fidelity 2X PCR Master Mix (2x) [10 µL], ddH₂O up to 20 µL), mixing the samples by pipetting, and briefly spinning.
   2. Heat the PCR mix at 98 °C for 30 s, followed by 12-14 cycles of 10 s at 98 °C and 20 s at 63 °C, and one cycle of 2 min at 72 °C.
   3. Purify the PCR products with 18 µL of paramagnetic beads (0.8x volume) into 20 µL of low-EDTA buffer.
   4. Measure the library concentration and fragment size distribution using high-resolution electrophoresis systems.
   5. Pool an equal amount of individual libraries, including the input, H3K4me3 ChIP, BrdU IP, and eSPAN samples, and perform parallel paired-end sequencing with the Illumina sequencing platform.

3. eSPAN Data analysis for yeast cells (1 day)

NOTE: This data analysis process applies only to the yeast data; the eSPAN data analysis for ES cells can be followed as outlined in a recent publication²³.

1. Extract raw sequence files, trim adaptor sequences by Trim Galore tools, and remove reads smaller than 10 bp to reduce the size of the output file and to avoid crashes of alignment programs. Ensure files are in the format file1_1.fq, file1_2.fq for paired reads:
   /TrimGalore-0.6.10/trim_galore file.fq-o trim --length 10 - paired
2. Generate bowtie index files for the reference genome:
   bowtie2-build genome.fa
3. Map the reads to the corresponding reference genome (Saccharomyces genome (sacCer3) for yeast) using Bowtie 2:
   bowtie2 -x genome -1 trim_1.fq -2 trim_2.fq --local --no-dovetail --no-discordant --maxins 1000 -D 20 -R 3 -N 1 -L 20 -i S,1,0.50
4. Remove multimap reads with SAMtools and duplicate reads with Sambamba tools:
   samtools view -bhs - q 40 input.bam -o output.bam
   sambamba-1.0.1.1-linux-amd64-static markdup output.bam filtered.bam -r
5. Use deeptools to generate the bigwig files of read coverage on Watson and Crick strands separately for each sample from clean bam files:
   bamCoverage -b filtered.bam -o watson.bw --filterRNAstrand reverse -bs 1 - extendReads
   bamCoverage -b filter.bam -o crick.bw --filterRNAstrand forward -bs 1 - extendReads
6. Calculate the average bias at each nucleosome position²⁵ for each sample based on the formula: log2 the forward (Watson strand)/ reverse (Crick strand) reads following the reference nucleosome positioning were separated²⁵. R scripts for these calculations are available at https://github.com/clouds-drift/eSPAN-bias. For a standard calculation, use the following commands. 
   NOTE: This step applies only to the yeast data; the bias calculation for ES cells can be followed as outlined in a recent publication²³.
   Rscript cal_bias.R -w watson.bw -c crick.bw -r replication_origins.bed
7. If necessary, adjust the strand bias by the BrdU bias:
   Rscript normalize_bias.R -i matrix_files -c classes_list -C condition_list --target target_list --control control_list
   NOTE: "matrix_files" is a list of files generated by the cal_bias.R script separated by commas. e.g. 'file1','file2','file3'. "classes_list" is a list of sample classes corresponding to each file separated by commas (e.g., 'brdu', 'H3K4me3_eSPAN', 'H3K56ac_eSPAN'). "condition_list" is a list of sample conditions corresponding to each file and separated by commas. Each file will be paired by class and condition for bias adjustment. "target_list" is a list of classes taken from the classes_list. It will define the classes that will act as the data to be adjusted by the controls (e.g., 'H3K4me3_eSPAN','H3K56ac_eSPAN'). "control_list" is a list of classes taken from the classes list. It will define the classes that will act as the data to be adjusted by the controls. Ensure that the list length is the same as the target_list (e.g., 'brdu')
8. After calculation and adjustment, group samples and create visualizations of the data.
   Rscript draw_bias.R -s' bias_matrix/file1.txt, bias_matrix/file2.txt' -o 'bias.pdf' -g group 1,group 2 --interval=T
   -s requires a list of bias files generated by the above scripts
   -o is the output file
   -g defines the experimental groups to calculate i.e., biological replicates to plot the average bias.
   --interval T or F, to display standard errors on the plots within experimental groups.
   NOTE: The information about replication origins was defined by BrdU IP data. The information of single nucleosome position was obtained from a previously published article²⁵. For the analysis of mouse ES cell data, the origin information was obtained from the OK-seq dataset²⁶, and the average bias calculation should follow the protocol outlined in previous work²³.

Resultados

eSPAN can be utilized to acquire strand-specific information regarding a target protein, such as histones and histone modifications. In this representative result, sample collection and eSPAN processes were conducted following the schematic outlined in Figure 2 using the budding yeast system. The sample treatment and collection follow the hydroxyurea treated early S phase condition as this protocol. Two yeast strains, WT (wild-type) and mcm2-3A mutant, were employed. The DNA helicas...

Discussão

Detection of proteins associated with leading and lagging replication strands is important for understanding the mechanisms of chromatin replication and the associated process, which are directly relevant to human cancer research and the mechanisms of drug resistance. Several methods, such as 2D gel, native Chromatin immunoprecipitation(nChIP), iPOND, and nascent chromatin capture^29,30,31, have been employed to study DNA replica...

Materiais

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH 8.0	Thermo Fisher Scientific	15575020
1.5 mL DNA low-binding tubes	Eppendorf	22431021
10x Ampligase buffer	Lucigen	A1905B
10x PBS	Corning	21-040-CV
10x TBS	BioRad	1706435
16 G x 1-1/2 in. BD PrecisionGlid Needle	BD Biosciences	305198
ACCEL-NGS 1S PLUS DNA LIBRARY KIT	Integrated DNA Technologies	10009817	ssDNA and low-input DNA library prep kit
Agilent 2100 bioanalyzer	Agilent	2100 bioanalyze
Agilent DNA 1000 kit	Agilent	5607-1504
Ampligase	Lucigen	A0110K	Handle paraformaldehyde with care, it is flammable solid hazardous.
AMPure XP	Beckman	A63881
AMPure XP SPRI Reagent	Beckman Coulter	A63882
Anti-BrdU antibody	BD Biosciences	555627
Anti-H3K4me3 antibody	abcam	ab8580
Automated cell counter , model TC10	BioRad	145-0001
Bacitracin	Sigma	11702
Bacto peptone	Thermo Fisher Scientific	211830
Benzamidine Hydrochloride	Sigma	B2417	Combustible Solids
BrdU	Sigma	B5002	Handle BrdU with care as it is a potential mutagen and teratogen.
BSA	New England Biolabs	B9000S
CaCl2	Sigma	C4901
Cell culture incubator	Thermo Fisher Scientific  model Forma series II water-jacketed CO2 incubator	3140
Cell culture medium
Cell line ES-E14
Chelex-100	BioRad	1422842	200–400 dry mesh size
Chloroform	Sigma	366919
Concanavalin A (ConA)-coated magnetic beads	Polysciences	86057-10
Digitonin	Millipore	300410-5MG
DMSO	Sigma	D8418
E. coli tRNA	Sigma	10109541001
Ethanol	Thermo Fisher Scientific	BP2818100	Handle ethanol with care, it is flammable
glucose	Sigma	G7021
Glycerol	Sigma	G5516
HCl	Sigma	H1758
HEPES	Sigma	H3375
Isopropanol	Thermo Fisher Scientific	BP26324	Handle isoproponol with care, it is flammable
KAPA HiFi HotStart Ready Mix	Roche	7958889001
KCl	Sigma	P9541
KOH	sigma	221473	Handle KOH with care, it is corrosive
Low-speed vacuum
Magnet stand	Millipore	LSKMAGS08
Mastercycler Gradient PCR Thermal Cycler	Eppendorf	5331
MgCl2	Sigma	M8266
Micrococcal nuclease  (MNase)	Worthington	LS004798
MinElute PCR purification kit	Qiagen	28006
Mini-beads beater	Mini BeadBeater 24 Disruptor	607
Mini-centrifuge	Eppendorf	5424R
MnCl2	Sigma	244589
Multiplatform shaker	Fisherbrand	02-217-765
Multiplatform shaker incubator	Benchmark	INCU-SHAKER 10L
Multithermal heat block	Eppendorf	Thermomixer C
N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS)	Sigma	T5130	200–400 dry mesh size
NaCl	Sigma	S9888
NaOH	Sigma	S8045	Handle NaOH with care, it is corrosive
NEBNext HiFi 2× PCR master mix	New England Biolabs	M0541
NP-40 Igepal CA-630	Thermo Scientific	J61055.AP
Paraformaldhyde	sigma	158127	Handle paraformaldehyde with care, it is flammable solid hazardous.
pA-Tn5 enzyme	Addgene	121137
Pefabloc	Sigma	11429868001
Phenol-chloroform-isoamyl alcohol	Invitrogen	15593049
Pipette P1000, P200, P20 and P2	Eppendorf	2231300004
PMSF	Sigma	10837091001
Protease inhibitor cocktail tablet	Sigma	S8830
Protease inhibitor cocktail tablet	Sigma	S8830
Protein G-sepharose beads	GE Healthcare	17-0618-02
Protein precipitation solution	Qiagen	848023
Protein precipitation solution	Qiagen	848023	Handle KOH with care, it is corrosive
Protein precipitation solution	Qiagen	848023
Proteinase K	Sigma	311584401
QIAquick PCR purification kit	Qiagen	28106
RNase A	Sigma	10109169001
SDS	Sigma	L3771
Spectrophotometer	Thermo Scientific.	2000/2000c
Spermidine	Sigma	S2626
T4 DNA Pol	New England Biolabs,	M0203
Table top centrifuge	Beckman Coulter	Allegra X-5
Tris	Sigma	11814273001
Triton X-100	Sigma	T8787
yeast extract	Thermo Fisher Scientific	212730