Guideline for bulk ATAC Sequencing
GENERAL GUIDELINES FOR SAMPLE PREPARATION
The ATAC-seq assay reveals the accessible regions of the chromatin in its native state, providing valuable information about the regulatory landscape of a cell type or tissue. For a successful ATAC-seq experiment, it is crucial that nuclei are fully intact.
CHECKLIST CRITICAL POINTS
The quality of nuclei isolation highly depends on tissue/cell types, thus, this step should be optimized to yield nuclei with the best quality possible. Because of specificities of each tissue type, we don’t perform the isolation of nuclei and the tagmentation reaction on site. Nevertheless, we are happy to provide general guidelines on sample preparation and on the tagmentation reaction itself.
Nuclei isolation
Optimization of lysis conditions is important to obtain intact nuclei. If tissue/cells are over lysed, nuclei can get disrupted and chromatin will leak out. This will result in a more homogeneous DNA fragmentation (not the wave-like nucleosomal profile) as chromatin won’t be in its native state anymore.
In general, we recommend being as gentle as possible to avoid over-lysing. Lysis times, detergent dilutions, types of mechanical dissociation (if needed), and time/speed of spinning of the dissociated nuclei solution are some of the most relevant variables that can affect nuclei isolation. One should carefully test these conditions prior to running the experiment, and keep in mind that different tissue/cell types need to be optimized independently.
A good quality control for the isolation is a visual inspection of the nuclei under a microscope (minimum 40x objective), after staining an aliquot with DNA-intercalating dyes such as propidium iodide, or vital dyes such as trypan blue. Nuclei should have a roundish shape, nuclear membrane should be intact and DNA should not be leaking out. Ideally, the solution should be fairly clear of cell debris. To obtain a more pure nuclei solution, it is possible to perform low-speed density gradient centrifugation (e.g. Percoll, sucrose or iodixanol gradients).
Tagmentation
There are a variety of published protocols providing guidance for the tagmentation reaction (see references below). The tagmentation reaction (enzyme and buffer concentrations) should also be optimized according to the number of nuclei used, to yield an ideal tagmentation profile. We recommend using the Illumina chemistry (Illumina Tagment DNA TDE1 Enzyme and Buffer, Cat.-no.: 20034197 or 20034198). We can provide aliquots of these reagents upon request.
After tagmentation, the reaction should be purified (e.g. QIAGEN MinElute Reaction Cleanup Kit or Beckman Coulter 1.4x XP beads) and can then be stored at -20°C.
INPUT MATERIAL
We can receive tagmented and purified DNA as well as ready-to-sequence libraries.
Tagmented DNA
In general, we prefer to receive purified tagmented DNA and perform the amplification and library indexing ourselves. This gives us the flexibility to choose barcodes that are compatible with other samples being processed simultaneously. When submitting tagmented DNA, please purify the reaction and elute the sample in 10µl DNase-free water or EB buffer. The samples do not need to be quantified.
Once we perform sample QC, we will reach out to discuss sample quality before proceeding with sequencing.
Ready-to-sequence libraries
These need to be dual-indexed with Illumina compatible barcodes. Please contact us before planning your experiments, so we can advise on the best barcoding options.
We will check the fragment size distribution of your samples on a capillary electrophoresis device. If fragments are larger than 900bp, or adapter dimers are detected, we will perform a size selection/purification step before loading the samples on the flowcell.
SEQUENCING
Standard sequencing for ATAC-Seq is paired-end 2 x 100bp. Different read lengths can be requested.
We recommend a sequencing depth of 60mio read pairs/sample. At least 3 biological replicates are needed for a proper data analysis. Specificities of the species, sample, or project design, can influence the optimal sequencing coverage. We are glad to provide guidance with this.
BIOINFORMATICS ANALYSIS
We have an established pipeline that generates both quantitative and qualitative information about the samples. The workflow comprises all steps from initial quality control, alignment to reference genome, peak calling, annotation of peaks relative to genes, and differential accessibility analysis. For detailed information about the pipeline and analyses, please see https://github.com/nf-core/atacseq.
SAMPLE SUBMISSION
Preparing aliquots for sequencing
When submitting tagmented DNA, please purify the reaction and elute the sample in 10µl DNase-free water or EB buffer. The samples do not need to be quantified.
Finalized ATAC-Seq libraries need to be uniquely dual-indexed with Illumina compatible barcodes. Please contact us before planning your experiments, so we can advise on the best barcoding options.
Supported plastics for sample submission
Samples can only be submitted in 96-well plates, 384-well plates or 8-strip PCR tubes with single lids. Strips or plates that are fully compatible with our systems are Sarstedt strips (#72.991.002), Eppendorf 96-well twin.tec® skirted PCR plates (e.g. Cat.no. 0030128648) and 4titude 384-well FrameStar plates (Cat. no. 4ti-LB0384/C). These can be provided upon request. When submitting in plates please fill them column-wise w/o empty wells in between.
Please identify tubes/samples with clear and short labels, e.g. with the sample ID (created in Rosalind during online sample submission) or a running index clearly referenced under the corresponding samples submitted over the Rosalind Client Portal.
Providing project and sample information via the Rosalind Client Portal
Please log in to Rosalind to enter all necessary project information making use of the “Project Description” field during the project set-up dialogue. Give all relevant details about project design, sample preparation method and how the samples are eluted and/or preserved. Sample-specific information such as concentration, volume, used preparation protocols etc. should be provided when the samples are added to the created project.
Please also indicate if you require a bioinformatics analysis during project setup.
Submission of large sample batches
The Rosalind customer portal offers an Excel-based alternative for sample submission of more than 30 samples delivered in either 8 well strips, 96- or 384-well plates. You can specify these supported formats in the portal’s sample submission dialog and download a formatted Excel template for entering your sample details. Plate identifiers and well coordinates are pre-set to facilitate easy data entry. Once completed, please email the Excel file to genomecenter@tu-dresden.de. Do not upload individual samples to Rosalind in this case.
To submit samples, you must be registered in our Rosalind client portal. Therefore, please send an e-mail to genomecenter@tu-dresden.de with your contact details (name, working group, working group leader, your e-mail address and telephone number). This will enable us to set up an account for you. After you have received an automatic e-mail, please log in to Rosalind with your login details. If needed, we can reset your login details.
Delivering or shipping of samples
Once the project and sample information has been entered into the Rosalind Client Portal, please put the samples in a small plastic bag labeled with your name and Rosalind project ID.
Samples can be personally delivered to the lab at the (CRTD, 3rd floor south), daily from 9-12h and 13-16h. If needed, contact us to deliver samples outside of this time window (+49 351 458 82362 or email us at genomecenter@tu-dresden.de).
Please deliver samples under suitable conditions! When shipping your samples, please use an adequate-sized box and ensure a proper amount of cooling material to hold the sample temperature until it reaches us. The package should be addressed to:
DRESDEN-concept Genome Center
c/o Center for Regenerative Therapies Dresden
Technische Universität Dresden
Fetscherstraße 105
01307 Dresden
Following up project progress
You can accompany the status of your project in our Rosalind Client Portal. Documents with information about the preparation method and quality control of samples and libraries are provided for your project.