ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

This modified ATAC-seq protocol introduces a new lysis buffer that decreases contaminating mitochondrial reads from 50%to less than 3%The decreased mitochondrial contamination will allow researchers to perform more efficient ATAC-seq with a 50%decrease in sequencing costs. We have validated this new ATAC-seq protocol in human primary PBMCs, human primary monocytes, mouse dendritic cells, and melanoma cell lines. We believe it will be effective in a wide range of cell types.

Minimizing sample loss during the cell activation and selection process is critical for obtaining high-quality results with ATAC-seq. We will demonstrate how to handle small numbers of cells from precious samples using basic laboratory equipment. To begin, thaw one vial of previously isolated CD4+T cells in a 37-degree Celsius water bath until the ice has just melted.

Then gently transfer the cells to a 15-milliliter conical tube containing nine milliliters of pre-warmed complete RPMI. Centrifuge the cells at 1, 500 times G, and four degrees Celsius, for six minutes. Then discard the supernatant, and gently resuspend the pellet in two milliliters of complete RPMI.

After repeating the centrifugation, discard the supernatant, and gently resuspend the cells in 0.5 milliliters of complete RPMI. Use a hemocytometer to count the cells. Using complete RPMI, adjust the cell suspension density to plate 50, 000 CD4+cells in 200 microliters per well of a 96-well round-bottom plate.

Then, to prepare magnetic beads, aliquot 12.5 microliters of Human T-Activator CD3/CD28 beads per ATAC-seq sample to a 1.5 milliliter tube. Add one milliliter of 1x PBS to the tube, and place it on a magnet for one minute to wash the beads. Carefully remove and discard the clear supernatant, and remove the tube from the magnet.

Resuspend the beads in 13 microliters of complete RPMI per ATAC-seq sample. To activate the CD4+T cells with the adjusted concentration, collect 500, 000 cells in 2.1 milliliters of complete RPMI medium in a 15-milliliter conical tube. Add 12.5 microliters of Human T-Activator beads, prepared and washed in previous steps, and gently mix by inverting the tube.

Then gently transfer the cells, with beads, to a sterile reservoir. Using a multi-channel pipette, plate 200 microliters of cells with beads per well of a round-bottom 96-well plate, and incubate in a 37-degree Celsius, 5%CO2 incubator for 48 hours. After complete incubation, centrifuge the plate at 423 times G, and room temperature, for eight minutes.

Remove 100 microliters of medium from each well, and collect it in a conical tube before discarding, in order to confirm no bead loss. Then resuspend the cell pellet in the remaining 100 microliters of medium, and collect everything in a 1.5-milliliter tube. To isolate CD4+cells, add 50 microliters of pre-washed CD4 conjugated beads to 500, 000 cells in one milliliter of complete RPMI, and invert to mix.

Incubate for 20 minutes at four degrees Celsius, while hand-flicking every six minutes to mix. Place the tube on the magnet for two minutes, until supernatant is clear, and then remove and discard. Remove the tube from the magnet.

Wash the bead-bound cells by resuspending them in one milliliter of PBS, plus 2%FCS, and place the tube back on the magnet for one minute. Discard the clear supernatant, and repeat this process for a total of three washes. After the final wash, place the tube on the magnet for one minute.

Remove it, discard the supernatant, and place the pellet on ice. Next, to perform nuclei isolation, resuspend the beads and cells in 50 microliters of cold lysis buffer, and centrifuge immediately at 500 G, four degrees Celsius, for 10 minutes. Remove and discard the supernatant.

To perform the transposase reaction, resuspend the isolated nuclei pellet with 50 microliters of Tn5 transposase mix. Incubate in a thermocycler for 30 minutes at 37 degrees Celsius, with the lid set to 40 degrees Celsius, and then hold at four degrees Celsius when complete. Immediately after thermocycling, perform a quick bench-top centrifuge spin-down.

Place the tube on the magnet for one minute to remove the beads from the product. Without removing the tube from the magnet, transfer the clear supernatant to a DNA purification column. Wash the column once with 250 microliters of PB buffer, and twice with 750 microliters of PE buffer.

Then add 10 microliters of elution buffer to elute the sample. Set up the initial PCR amplification reaction in a nuclease-free PCR tube. Place the PCR tube into the thermocycler, and run the PCR amplification program.

Then complete the final PCR amplification of the remaining 45 microliters of PCR reaction, as described in the manuscript. Place the PCR tube in the thermocycler and run the program. After complete amplification, purify the libraries using a PCR cleanup kit, following the manufacturer’s protocol, eluting in 25 microliters of the elution buffer.

Sequence the prepared libraries on a next-generation sequencer to an average read depth of 42 million reads per sample. This improved ATAC-seq protocol produced a final library of greater than one nanogram per microliter for sequencing. Quality control showed DNA fragments between 200 and 1, 000 base pairs, and further sequencing was performed with these high-quality libraries.

Libraries prepared following this protocol showed only 3%mitochondrial contamination. The high percentage of usable reads is sufficiently constant across biological replicates. This protocol provided highly reproducible results across technical replicates, as well as biological replicates.

Additionally, this protocol took 48 hours, rather than one week or more as previously described, resulting in consistent and efficient activation, as demonstrated by reproducible sequencing results. Furthermore, predicted ATAC-seq peaks were accurately called by the analysis pipeline. Sequencing result analysis revealed clear changes in chromatin state during human T cell activation.

Differentially accessible regions of open chromatin were identified between six samples before and 48 hours after activation. Make sure the nuclei lysis reaction is performed with chilled reagents and materials, in order to maximize the recovery of intact nuclei. Our modified nuclei lysis buffer is gentler on the cells, and minimizes contaminating mitochondrial DNA.

We look forward to this modified lysis buffer being applied to single-nuclei ATAC-seq as well. We know this protocol will allow researchers to produce higher-quality ATAC-seq data at a decreased cost, helping to forward the epigenetic field. The protocol is straightforward, rapid, and does not require any hazardous reagents or instruments, making it easily accessible for those new to ATAC-seq.