The overall goal of this high-throughput method using transposase is to identify accessible chromatin regions in the human genome. This method can help answer key questions in the epigenomic fields such as genome-wide locations of regulatory elements. The main advantages of this technique are that it is robust, relatively short and requires less starting material relatively to other methods of measuring chromatin accessibility such as DNase-seq or FAIRE-seq.
Though this method can provide insight into regulatory landscape of human T lymphocytes, it can also be applied to other systems such as other human primary cells, cancer cells, human clinical samples, cells from other mammals and organisms. Thaw a one-milliliter aliquot of 10 million human PBMCs and transfer it to a 50-milliliter tube containing 10 milliliters of supplemented RPMI medium. Them, centrifuge the cells at 500 G for five minutes at four degrees Celsius.
Next, remove the supernatant and resuspend the cells in 15 milliliters of supplemented RPMI medium. Then, transfer the cells to a T-75 culture flask and incubate them overnight with humidification. The next day, transfer the floating cells with a sterile 25-milliliter pipette to a 50-milliliter tube.
Then, count the living cells using trypan blue exclusion. Next, isolate the CD4 positive cells from 10 million living, non-adherent PBMCs using a microbead separation column. Once the cells are isolated, plate and activate the T cells in Th1 and Th2 polarizing conditions according to the text protocol, and then isolate their nuclei.
To being, prepare fresh lysis buffer and keep it chilled on ice. Also, in preparation for the transposition reaction, warm a thermal shaker to 37 degrees Celsius. Next, load a half million T cells into 1.5-milliliter microtubes and spin them down at 500 Gs for five minutes at four degrees Celsius.
Next, wash the cells with one milliliter of cold PBS and repeat the spin cycle, but now suspend the cells in one milliliter of cold lysis buffer. Mix the cells with gentle pipetting to prevent the nuclei from being disrupted too much. Then, quickly take 10-microliter aliquots to observe the fraction of cells'lyse.
Be sure to keep the cells cold and work quickly. Within five minutes, proceed with the transposition reaction. To begin, transfer 100, 000 nuclei to a 1.5-milliliter microtube and spin down the sample at 500 Gs for 10 minutes in a cold centrifuge.
Then, gently remove the supernatant. Next, add the transposition reaction components to the nuclei. Then, resuspend the nuclei with gentle pipetting and incubate the nuclei on the thermal shaker at 500 RMP for 30 minutes to complete the transposition reaction.
Next, perform a DNA cleanup step and elute the DNA fragments in 20 microliters of Tris hydrochloride. Then, use a PCR to make an initial amplification of the ATAC-sequencing libraries. Next, assess the optimal number of additional cycles needed for the final amplification by quantitative PCR.
From the measured results, determine the number of cycles needed for the final PCR amplification of the ATAC-sequencing libraries. Then, perform the final amplification. Setting up an optimal size of DNA library fragments can improve the next generation sequencing.
First, warm up the magnetic beads to room temperature and prepare fresh 70%ethanol in nuclease-free water. Next, add nuclease-free water to the ATAC-sequencing libraries to a volume of 100 microliters. Then, add 50 microliters of resuspended DNA binding magnetic beads.
Pipette the beads into the DNA at least 10 times and wait five minutes. If any drops of beads get stuck on the tube wall or lid, briefly spin the tube. Now, place the sample next to the magnet for two minutes, and then transfer the supernatant to a new microtube.
Measure the volume of the collection and add a 70%volume of magnetic bead suspension. Mix the beads in as before and let them incubate for five minutes before proceeding. Then, separate the beads using the magnet for two minutes and now discard the supernatant.
While still against the magnet, add 200 microliters of 70%ethanol to the beads. Wait 30 seconds then discard the ethanol and repeat the ethanol wash once more. Finish the washes by completely removing the remaining ethanol and letting the beads air-dry for five minutes on the magnet.
If necessary, briefly spin the tube and aspirate out any visible ethanol with a 10-microliter pipette. Now, remove the tube from the magnet and add 22 microliters of Tris hydrochloride. After two minutes, return the tube to the magnetic stand and let the solution clear.
Then, collect 20 microliters of eluate containing the prepared library and store it at minus 20 degrees Celsius. This protocol produces an ATAC-sequencing libraries that are typically three to 20 nanograms per microliter. When run on a system for DNA integrity analysis, they have a ladder-like appearance.
Amongst others, another important quality control is the enrichment of the positive controls. Relative to the negative controls, primer pair three should be enriched at least 25-fold, and primer pair four should be enriched at least 75-fold. After an initial 10 million reads, if the sample passes all crucial parameters in FastQC Report file, and 1, 000 to 2, 000 peaks are obtained from peak calling, then the library can be sequenced more deeply for greater than 30 million reads.
From libraries meeting the quality standards, only six to 20%of the reads map to the mitochondrial genome. The remaining unique reads map to the reference human genome and seven to 12%are within the ATAC-sequencing peaks. After watching this video, you should have a good understanding of how to make ATAC-seq libraries, submit and furnish ration sequencing and to analyze the obtained data.
Once mastered, the ATAC-seq library preparation can be done in one or two working days. While attempting this procedure, it is important to remember that you will maybe need to first optimize some steps like the number of the cells and the composition of the lysis buffer in a nuclei isolation step. Following this procedure, other methods like chromatin immunoprecipitation can be performed in order to answer additional questions like which transcription factor binds to the open chromatin regions in the examined samples?
