A method to identify open chromatin regions, such as promotor areas, using a transposase.
ATAC-seq works by incorporating primer sequences into chromatin using a TN5 enzyme (tagmentation). Areas in the chromatin that are densely packed, for example in heterochromatic regions, will prevent the incorporation. The primer sequences will thus only be added to open, euchromatic areas. One typical region containing open chromatin is the regulatory area of gene promoters.
Omni-ATAC is an improved ATAC-seq protocol for chromatin accessibility profiling. The Omni-ATAC protocol lowers sequencing costs by generating fewer sequencing reads that map to mitochondrial DNA, consequently a higher percentage of reads map to peaks of chromatin accessibility.
The Omni-ATAC method as set up at NGI can only be used for freshly-isolated cells, frozen nuclei pellets or tagmented DNA (prepared using the Omni-ATAC protocol provided by NGI).
- Sample type:
- freshly isolated. Viability >90%.
- counted and resuspended in media.
- snap-frozen pellet.
- preferably obtained using sucrose sedimentation.
- Tagmented DNA
- purified DNA fragments after the tagmentation reaction performed by the user following NGI’s protocol.
- Sample amount:
- Cells or nuclei: 50 000 per tube for a ~3Gbp genome organism.
- Tagmented DNA: result from NGI’s tagmentation protocol.
- Sample delivery:
- Cells: delivered on ice at a time agreed upon with the responsible lab personnel at NGI.
- Nuclei: snap frozen pellet, on dry ice. Can be sent by post and the package should be labeled as “Cold Delivery, –20˚C”
- Tagmented DNA: can be sent by post and the package should be labeled as “Cold Delivery, -20˚C”
Although the protocol allows for some margin of error, the input amount should be kept the same across samples. Starting with too few cells may cause the library prep chemistry to fail due to insufficient DNA input, whereas too many will result in a higher than optimal DNA-to-tagmentase ratio. This generates longer DNA fragments, which perform poorly in sequencing. You are therefore required to count the cells or nuclei before submission.
For samples from sources with significantly larger or smaller genomes, the number of cells needs to be adjusted accordingly. Please ask your project coordinator for details if the samples are of non-mammalian origin.
How to evaluate the sample quality
We cannot check the correct amount of cells/nuclei or the quality of your samples. You should thus make sure of the following:
- count the cells/nuclei as accurately as possible.
- don’t deliver more than 50.000 cells/nuclei per sample (or other amount, if discussed with NGI)
- make sure that your counted cells are alive – a high proportion of dead cells in the sample will reduce data quality.
If you are not able to carry out these steps, or your samples are below the required thresholds, please get in touch.
What we do with your samples
When receiving cells from cell culture, we first spin down the cells and then resuspend the cells in a mild lysis buffer to generate a crude nuclei extract. The nuclei then are spun down and the transposition reaction is added directly.
The Omni ATAC protocol includes the use of multiple detergents for cell lysis to improve permeabilization and remove mitochondrial DNA. For the tagmentation reaction, the TN5 enzyme is used to simultaneously fragment DNA and insert primers into the cut site. The chromatin is subsequently cleaned up using DNA purification columns. At this stage, the tagmented DNA has single-stranded overhangs due to the primers added during the tagmentation reactions. These are repaired by a single elongation step prior to the first PCR amplification with 5 cycles.
In order to amplify the libraries as little as possible, the number of additional cycles is determined by subjecting an aliquot of the PCR reaction to a qPCR. The number of cycles is chosen where the amplification signal reaches 1/4 of the final signal level.
After the library is further amplified by the additional cycles, the final library is purified using AMPure XP beads and the concentration and fragment sizes are determined.
Library QC and sequencing
In this step, we evaluate the yield obtained and the size distribution of the libraries. We will inform you of the QC status of each sample. Once the libraries have passed this QC step, they are queued for sequencing, to be carried out according to the setup stated in the agreement.
We have access to 96 (combinatorial) dual indexes so that is currently the highest available level of multiplexing. The optimal sequencing depth depends on genome size and level of open chromatin, so for samples where these are expected to be very different from the typical sample, the sequencing depth may need to be adjusted. We typically recommend 50 million paired-end sequence reads per library for human samples.
We expect more sequencing reads from euchromatic regions than from heterochromatic regions. Areas with open chromatin conformation – eg. the transcriptional start sites of active genes – are also expected to get more reads. Results will furthermore depend on the characteristics of the samples and how they are sequenced:
- Paired-end sequencing is required when information on nucleosome positioning is of interest.
- For general information on chromatin status changes, single-end sequencing can be sufficient.
- Duplicate detection is significantly improved with paired-end sequencing.
- If too much DNA was provided, the fragment length distribution of the library will be shifted towards longer, high molecular weight fragments. We do not recommend the sequencing of such samples.
- If the nuclei were lysed before submission, we would expect higher background noise levels in the sequencing data.
The bioinformatics analysis that we provide (if available for your species of interest) for ATAC-seq is based on the nf-core/atacseq pipeline. It provides alignment, QC, filtering, peak calling, normalization, and differential accessibility analysis. Read more about the pipeline output here: https://nf-co.re/atacseq/docs/output.
References and links
- An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues
- ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide
- nf-co.re/atacseq analysis pipeline
Last Updated: 22nd January 2024