Guideline for bulk ATAC Sequencing
GENERAL GUIDELINES FOR SAMPLE PREPARATION
The ATAC-seq assay identifies accessible regions of chromatin in its native state, providing insights into the regulatory landscape of cells or tissues. For a successful experiment, fully intact nuclei are essential.
CHECKLIST CRITICAL POINTS
Nuclei isolation
The quality of nuclei isolation depends on tissue and cell types, why we don’t perform the isolation of nuclei and the tagmentation reaction on site. Optimization of lysis conditions is crucial to yield intact nuclei. Over-lysis can disrupt nuclei and result in non-native chromatin states, leading to homogeneous DNA fragmentation instead of the characteristic nucleosomal profile.
Key factors for optimization include:
- Lysis conditions: Test lysis times (e.g., 5 and 10 minutes) and detergent dilutions (e.g., 0.1%, 0.05%, 0.01%).
- Mechanical dissociation: Adapt type, duration, and speed as required.
- Centrifugation: Optimize time and speed for better results.
If enough cells are available, conduct a pilot experiment using ∼30,000 cells per condition to determine optimal lysis parameters. A good quality control involves staining nuclei with DNA-intercalating dyes (e.g., propidium iodide), or vital dyes such as trypan blue and visually inspecting them under a microscope (40x objective). Intact nuclei should appear round, with no DNA leakage and minimal debris. If necessary, use low-speed density gradient centrifugation for purification (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). Aliquots of these reagents can be provided upon request.
For full flexibility of the sequencing run planning we prefer the submission of tagmented DNA samples for subsequent indexing PCR and library preparation. The tagmented samples 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.
If you prefer to perform Indexing PCR yourself, ensure uniquely dual-indexed (UDI1) libraries with Illumina-compatible barcodes. Libraries with incompatible indexes must be run on separate flowcell compartments. Refer to these information for ordering indexing primers:
INPUT MATERIAL
We accept either purified tagmented DNA or ready-to-sequence libraries:
- Tagmented DNA: Purify and elute in 10 μl DNase-free water or EB buffer. Quantification is not required.
- Ready-to-sequence libraries: Ensure unique dual indexing with Illumina-compatible barcodes. Contact us for guidance on barcoding options.
We perform QC on all submitted samples. For ready-to-sequence libraries, we verify fragment size distribution. Libraries with fragments >900bp or adapter dimers will undergo size selection or purification before sequencing.
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
- Tagmented DNA: Purify and elute in 10 μl DNase-free water or EB buffer.
- Libraries: Ensure dual indexing with Illumina-compatible barcodes. Volume >= 10 ul and a concentration >= 10 ng/ul. When you obtain less material, please get in touch with us before submitting those samples.
- Plastics to use: we only accept those
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.
When submitting ready-to-sequence NGS libraries we need the comprehensive details about the library prep protocol, the indexing scheme and NGS construct design (TruSeq, Nextera, custom etc.).
For submitted NGS libraries, the library index combinations should be added to the “Notes” field for each individual sample. This is different between DcGC-supported and custom / commercial index sets.
Please provide the sequence or DcGC-supported name of Index in the Notes field. If you have any questions or feel unsure about an information, please contact us by email.
In case you are using commercial index sets, please provide comprehensive documentation by email. To avoid any misunderstanding we recommend to get in touch with us before starting to prepare - or latest before submitting - your samples. It will be essential to
- Email us a file with the index set name (supplier, item number) and index set sequences so it can be added to our laboratory database (Rosalind)
- Afterwards we will provide you the naming format of the indexes
- Use the exact same names for the index combinations, separated by semicolon, when adding the index information to the “Notes” field of each individual sample. You can add additional information to the notes field, they should be separated by semicolon from the index name
- The sequencing mode and Flowcell requirements are clearly discussed.
If you have any questions or feel unsure about an information, please contact us by email.
To submit samples, you must be registered in our Rosalind Portal. Therefore, please send an email to genomecenter@tu-dresden.de with your contact details (name, working group, working group leader, your email address and telephone number). This will enable us to set up an account for you. After you have received an automatic email, 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 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 Portal. Documents with information about the preparation method and quality control of samples and libraries are provided for your project.
REFERENCES
Footnotes
Unique dual indices (UDIs) minimize the impact of index hopping that can potentially occur during Illumina sequencing and results in the misassignment of reads to a different Illumina index. It uses unique identifiers on both ends of each Illumina NGS library.↩︎