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Assay for Transposase-Accessible Chromatin with High-Throughput Sequencing (ATAC-Seq) Protocol for Zebrafish Embryos

Experiment Summary

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a useful method to map genome-wide chromatin accessibility and nucleosome positioning. Genome-wide sequencing is performed utilizing adapter sequences inserted by a prokaryotic transposase, Tn5, into the accessible regions of chromatin. Here we describe the use of ATAC-seq in the zebrafish embryo and thereby the applicability of this approach in whole vertebrate embryos.

Materials and Reagents

A. Embryo Dissociation and Cell Preparation Components

  1. Tweezers.
  2. 100-mm petri dish.
  3. Incubator (28.5°C).
  4. 1.5-ml microfuges.
  5. 15-ml falcon tubes.
  6. Micro pestle.
  7. 1× E3 embryonic medium.
  8. 1× phosphate-buffered saline.
  9. Lysis buffer.

B. Transposition and PCR Amplification Components

  1. Heating block.
  2. Gradient PCR Instrument.
  3. Microcentrifuge.
  4. 0.2-ml PCR tubes.
  5. Fast 96-well reaction plate (0.1 ml).
  6. Optical adhesive.
  7. Real-Time PCR System.
  8. DNA Sample Prep Kit.
  9. Reaction Cleanup Kit.
  10. SYBR Green I nucleic acid gel stain.
  11. Next High-Fidelity 2× PCR Master Mix.

Procedure

A. Embryo Dissociation and Cell Preparation

  1. Collect zebrafish embryos in a 100-mm petri dish, and raise in 1× E3 embryo medium in an incubator set to 28.5°C until desired stage.
  2. Take a number of embryos with an approximate total cell number of 50,000.
  3. Collect dechorionated embryos in a 1.5-ml microcentrifuge tube and dissociate using a micro pestleand follow by mixing using a 200-μl pipette. Wash the pestle into the tube with 1× E3 embryo medium to collect all the cells on the pestle.
  4. Centrifuge immediately at 500 × g, 4°C for 5-10 min.
  5. Remove supernatant without disturbing the cell pellet. Add 50 μl of cold 1× PBS and centrifuge at 500 × g, 4°C for 5 min.
  6. Remove supernatant and add 50 μl of cold lysis buffer. Gently flick the tube and then centrifuge immediately at 500 × g, 4°C for 10 min. Place the tube on ice.

B. Transposition

  1. Prepare 50 μl of transposition mix per reaction combining 25 μl Tagment DNA buffer, 2.5 μl Tagment DNA enzyme 1, and 22.5 μl nuclease-free H2O. Add this to the lysed cells and gently mix by pipetting.
  2. Incubate at 37 °C for 30 min on a heating block.
  3. Clean reaction using Reaction Cleanup Kit. Perform centrifugations at room temperature and at 12,000 × g.
  4. Add 300 μl Buffer ERCto the reaction and mix. Place a MinElute column in a 2-ml collection tube, load the sample to the column, and centrifuge for 1 min. Empty the collection tube.
  5. Add 750 μl Buffer PE to the MinElute column and centrifuge for 1 min. Empty the collection tube.
  6. Centrifuge for 2 min to remove residual ethanol from the PE buffer.
  7. Place the MinElute column in a clean 1.5-ml microcentrifuge tube. Add 10 μl of elution buffer to the column, incubate for 1 min at room temperature, and centrifuge for 1 min to elute DNA. Purified DNA sample can be stored at -20°C at this point.

C. PCR Amplification

1. For PCR amplification, mix the reagents below in a PCR tube:

10 μl of nuclease-free H2O.

10 μl of transposed DNA.

2.5 μl of Nextera PCR Primer 1.

2.5 μl of Nextera PCR Primer 2.

25 μl of Next High-Fidelity 2× PCR Master Mix.

Run the PCR reaction:

72°C, 5 min

98°C, 30 s

5 cycles:

98°C, 10 s.

63°C, 30 s.

72°C, 1 min.

Hold at 4°C.

2. Determine cycle number using qPCR. Prepare 15 μl of qPCR master mix:

5 μl of 5 cycle PCR-amplified DNA.

3.9 µl of nuclease-free H2O.

0.25 µl of Nextera PCR Primer 1.

0.25 μl of Nextera PCR Primer 2.

0.6 μl of 100× SYBR Green I.

5 µl of Next High-Fidelity 2× PCR Master Mix.

3. Add reaction mix to a 96-well reaction plate and seal with optical adhesive film.

4. Run the qPCR reaction:

98°C, 30 s.

20 cycles.

98°C, 10 s.

63°C, 30 s.

72°C, 1 min.

Hold at 4°C.

5. Determine the number of cyclesto run for the remaining PCR reaction. For this, plot linear fluorescence vs. cycle in the StepOne software (Applied Biosystems), and make note of the cell-cycle number at which SYBR fluorescence intensity is half of the maximum fluorescence.

6. Return the rest of the PCR reaction back to the qPCR machine and run PCR reaction with the determined cycle number.

98°C, 30 s.

Use number of cycles as determined in step 6.

98°C, 10 s.

63°C, 30 s.

72°C, 1 min.

7. Clean reaction using Reaction Cleanup Kit.

8. Place the MinElute column in a clean 1.5-ml microcentrifuge tube. Add 25 μl of elution buffer to the column, incubate for 5 min at room temperature, and centrifuge for 1 min to elute DNA. Purified DNA sample can be stored at -20°C.

D. Sequencing and Analysis Considerations

  1. Sequencing depth. Sequencing depth. Low-depth sequencing consistently reveals the most accessible genomic regions but leaves weakly accessible regions undiscovered. However, it is currently impractical to generate enough sequence depth to fully saturate the entire dynamic range of genome-wide accessibility. Therefore, great care should be put into determining the depth of sequencing to be used for each replicate based on the goals of the particular experiment
  2. Replicates. Often the most difficult choice to make concerns the balance between number of biological replicates and sequencing depth. Many of the most accessible sites will be found in any cell line consistently. It is especially important for lower- accessible sites to have both enhanced coverage and enough replicates to assess biological variability.
  3. Paired-end vs. single-end reads. While paired-end sequencing is not necessary for establishing the locations of accessible chromatin and single-end sequencing will yield more coverage for the same cost, paired-end sequencing is helpful for identifying regions with more precisely positioned nucleosomes. The choice of whether to use single- or paired-end sequencing should be made accordingly.
  4. Read length. 50 bp reads map uniquely to the vast majority of the genome. Given the limitations of most sequencing budgets, it often makes more sense to increase depth of coverage than to increase sequence length, as mappability does not increase dramatically as reads get longer than 50 bp.
  5. Quality control. Once reads have been mapped to the genome of choice, for most projects, only fragments that map uniquely to the genome (mapq ≥ 30) should be used. To track quality, it is often useful to take note of the percent of tags that map uniquely to the genome in addition to the fraction of reads that represent mitochondrial "contamination."
  6. Integration location. The exact integration location represented by each read can be identified as the 5′-most position of reads that map to the reference strand +4 bp. For reads that map to the non-reference strand, the location is the 3′-most position of the read -5 bp.

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