ATAC-Seq Optimization for Cancer Epigenetics Research

ATAC-seq allows us to identify the status of the open chromatin regions in the genome, which is often altered in the disease states compared to the normal condition. Fewer cell input, a simplified protocol of Tn5 digestion, followed by isolation of fragmented DNA and library preparation by PCR amplification and shorter experimental time make it a more advantageous technique. Comparison of the altered epigenetic landscape by ATAC-seq between normal and disease conditions can shed light on the disease-associated chromatin regulatory elements and be useful for designing new therapeutic strategies.

This technique is easy to perform, as it does not include any critical experimental steps. To get successful ATAC-seq results, it only requires a couple of standardization steps. To begin, transfer 25 microliters from a cell suspension containing one million cells per milliliter in PBS to 1.7-milliliter microcentrifuge tube and centrifuge at 500 G for five minutes at four degrees Celsius.

Carefully discard the supernatant, and add 25 microliters of lysis buffer. Re-suspend the cells by gently pipetting up and down, and incubate on ice for five minutes. Centrifuge at 500 G for five minutes at four degrees Celsius, and carefully discard the supernatant.

Immediately re-suspend the pellet in 25 microliters of Tn5 reaction mixture, and incubate for 30 minutes at 37 degrees Celsius. Mix the solution every 10 minutes. Add five microliters of 3-molar sodium acetate and 125 microliters of buffer PB to the Tn5 tagmented DNA solution.

Mix well. Next, place the spin column in a 2-milliliter collection tube, and apply the sample to the spin column. Centrifuge at 17, 900 G for one minute at room temperature, and discard the flow-through.

Add PE buffer to the column, and spin the column. Place the spin column back in the same collection tube, and centrifuge it for five minutes to dry the membrane completely. Discard the flow-through, and place the spin column in a new 1.7-milliliter microcentrifuge tube.

Elute the DNA fragments with 10 microliters of buffer EB, and let the column stand for one minute at room temperature. Then centrifuge at 17, 900 G for one minute at room temperature to elute the DNA. Set up the PCR reaction in 200-microliter PCR tubes, and perform PCR Amplification Program Part One.

For real-time QPCR, prepare the PCR reaction mixture by adding five microliters of PCR product, 0.75 microliters of 1, 000-times-diluted SYBR Gold, five microliters of 2X PCR Master Mix and 3.75 microliters of nuclease-free water. Determine the required number of additional PCR cycles using the run parameters. Once the cycle number is determined, set up PCRs with the additional cycles calculated earlier.

To the amplified PCR products, add 150 microliters of beads, and incubate for 15 minutes at room temperature. Place the tubes on a magnetic stand for five minutes, and carefully remove the supernatant. Wash the beads with 200 microliters of 80%ethanol, and remove the supernatant.

Remove the ethanol completely, and air dry the samples for 10 minutes at room temperature. Finally, re-suspend with 50 microliters of elution buffer. Place the tube on a magnetic stand, then transfer the eluate to a fresh 1.7-milliliter microcentrifuge tube.

A comparison of cell lysis efficiency using trypan blue is shown here. NMuMG cells were treated with a hypotonic buffer and CSK buffer and stained with trypan blue. Higher cell lysis efficiency was observed in CSK-treated cells.

ATAC-seq libraries were prepared from MDA-MB-231 breast cancer cells. After PCR amplification, PCR products were analyzed on a 0.5X TBE, 1.5%agarose gel before and after beads purification. Primers are mostly removed by the initial beads purification.

DNA band patterns from 1X TAE 1%agarose gel electrophoresis and an automated electrophoresis are also indicated. SYBR Gold was used to visualize the DNA fragments shown here. A genome browser track showing the ERS1 locus is presented here.

The genome coverage of the ATAC-seq data in T47D cells is shown in this image. Primers from a previous publication were used, and their target regions are highlighted in yellow. A bar graph depicting primer amplicon enrichment in ATAC-seq libraries is shown here.

ATAC-seq libraries were prepared by the hypotonic buffer or a CSK buffer. The representative image shows the genome browser visualization for ATAC-seq optimization. The ATAC-seq data from the 25, 000-cell input condition showed the highest enrichment of nucleosome-free fragments, while the 100, 000-cell input condition presented the highest mononucleosomal DNAs.

Genome browser tracks showing ATAC-seq data from different cell numbers are presented here. HOMER peak annotation analysis is depicted in this representative image. HOMER classified each peak as a promoter proximal or distal peak.

The number of cell input choice of cell lysis buffer and the concentration of the Tn5 enzyme dependent on the cell type are the most critical parameter that must be standardized. Tn5 fused with protein A in the cut-and-tack technique is widely used to identify DNA binding site for a protein of interest using antibodies specific to that protein of interest.