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Fixed Cell ATAC-Seq Kit Overview
Need to perform ATAC-Seq on fresh or frozen cells or tissues? See our ATAC-Seq Kit.
Looking for recombinant Tn5 transposase? Click here.
Need to multiplex more than 16 samples? See our Nextera™-Compatible Multiplex Primers (96 plex).
ATAC-Seq is a rapid assay that allows analysis of epigenetic profiles across the genome by identification of regions that have open or accessible chromatin states. Because of the assay’s speed, simplicity, and applicability to a wide range of sample types, ATAC-Seq has become a commonly-used epigenetic assay, and it can serve as a gateway to further, more detailed epigenetic analyses.
In the Fixed Cell ATAC-Seq assay, intact nuclei from formaldehyde-fixed cell samples are treated with a hyperactive Tn5 transposase mutant. This transposase simultaneously tags the target DNA with sequencing adapters and fragments the DNA in a process termed tagmentation.
The Fixed Cell ATAC-Seq Kit from Active Motif provides the reagents necessary to produce 16 unique sequencing-ready Illumina®-compatible ATAC-Seq libraries from 50,000 – 100,000 formaldehyde-fixed cells per reaction. The optimized protocol guides you through the steps for sample preparation, tagmentation and library preparation, yielding next-gen sequencing-ready libraries that can be multiplexed in a single flow cell sequencing run.
Fixed Cell ATAC-Seq Kit Highlights:
- Compatible with formaldehyde-fixed cell samples
- Requires only 50,000 to 100,000 cells per reaction
- Simple and rapid optimized protocol & reagents
Fixed Cell ATAC-Seq Kit Contents
The Fixed Cell ATAC-Seq Kit is shipped at two temperatures, with one box on dry ice for components to be stored at -20°C, and a second box at room temperature for components to be stored at room temperature or 4°C. Please store components according to the storage conditions in the manual after first use. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents included:
- Hypotonic Buffer, store at RT
- Hypotonic Quench, store at 4°C
- Releasing Buffer, store at RT
- Proteinase K, store at -20°C
- RNase A, store at -20°C
- Protease Inhibitor Cockatil, store at -20°C
- Assembled Transposomes, store at -20°C
- 2X Tagmentation Buffer, store at -20°C
- 10X PBS, store at RT
- 10% Tween 20, store at RT
- 1% Digitonin, store at -20°C
- 0.5 M EDTA, store at RT
- DNA Purification Columns SF, store at RT
- DNA Purification Binding Buffer, store at RT
- DNA Purification Wash Buffer, store at RT
- DNA Purification Elution Buffer, store at RT
- 3 M Sodium Acetate, store at RT
- 10 mM dNTPs, store at -20°C
- 5X Q5 Buffer, store at -20°C
- Q5 High-Fidelity DNA Polymerase, store at -20°C
- i7 Indexed Primer 1, store at -20°C
- i7 Indexed Primer 2, store at -20°C
- i7 Indexed Primer 3, store at -20°C
- i7 Indexed Primer 4, store at -20°C
- i5 Indexed Primer 1, store at -20°C
- i5 Indexed Primer 2, store at -20°C
- i5 Indexed Primer 3, store at -20°C
- i5 Indexed Primer 4, store at -20°C
- SPRI Beads, store at 4°C
Fixed Cell ATAC-Seq Kit Data
The Fixed Cell ATAC-Seq kit contains the necessary reagents and enzymes to perform 16 ATAC-Seq reactions on formaldehyde-fixed cell samples. These kit components are optimized to work efficiently and have been validated to generate high-quality ATAC-Seq data.
Fixed Cell ATAC-Seq Results Compared with Native Cell ATAC-Seq Results
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Figure 1: Fixed Cell ATAC-Seq Compared to ATAC-Seq on Native Frozen Cell ATAC-Seq
Peak profiles are similar between Fixed Cells and Native Cells. Fixed Cell ATAC-Seq in general yields higher background results and lower peak intensity.
Fixed Cell ATAC-Seq is Compatible with 1,000 to 100,000 Formaldehyde-Fixed Cells
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Figure 2. Fixed Cell Titration ATAC-Seq Results
Duplicate samples of 1,000, 6,250, 12,500, 25,000, 50,000, and 100,000 formaldehyde-fixed K562 cells were assayed with the Fixed Cell ATAC-Seq kit. Higher background was observed with lower cell numbers, and peak profiles were consistent.
Formaldehyde Fixation Time from 3 to 15 Minutes of Fixation
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Figure 3. Fixation Time Titration of 50,000 K562 Cell Aliquots
Duplicate samples of 50,000 K562 cells were fixed with formaldehyde for 3,5, 7, 10, 12, and 15 minutes. Higher background was observed in cells fixed for less than 10 minutes.
Fixed Cell ATAC-Seq is Compatible with Formaldehyde-Fixed Sorted Blood Cells
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Figure 4. Genome browser tracks of sorted CD4+ cells from human blood samples compared with K562 cells and ENCODE CD4+ data.
Fixed Cell ATAC-Seq was performed on 200,000 formaldehyde-fixed sorted CD4+ human blood cells and 200,000 formaldehyde-fixed K562 cells. Similar peak profiles were observed betweeen the formaldehyde-fixed CD4+ blood cells and the ENCODE CD4+ data, and some differences in peaks were observed between the formaldehyde-fixed CD4+ blood cells and formaldehyde-fixed K562 cells.
(Click image to enlarge)
Figure 5. Genome Browser Results of Sorted and Formaldehyde-fixed Human B Cell Titration in Fixed Cell ATAC-Seq.
Sorted and formaldehyde-fixed B cells were run in duplicates of 50,000, 100,000, 150,000, and 200,000 cells. Genome browser data shown at the CD22 locus alongside ENCODE native human B cell ATAC-seq data.
Fixed Cell ATAC-Seq FAQs
What’s the best way to fix cells?
Please see the Kit manual for our recommended fixation protocol. Best results are obtained when fresh, methanol-free formaldehyde is used. We recommend 16% formaldehyde that is provided in an ampule such as Pierce™ 16% Formaldehyde (w/v), Methanol-free Catalog Number 28906 available from Thermo Fisher Scientific. If preparing your own Formaldehyde Solution, the formaldehyde must be added fresh, just before using the solution to fix your samples.
Does sonicating fixed cell samples improve results?
No. We did not observe improvement in data generated with the assay by sonicating fixed cell samples.
Why is there high background in the sequencing data?
Yes, high background is expected with fixed cells in ATAC-Seq. The background is always higher in fixed cells versus native cells. Please see reference 4, Zhang et al. 2022.
Why was no library produced?
Sample loss is very likely. Be sure to leave behind 10 µL of sample at removal of Hypotonic Buffer stage (step 11) and after the high speed spin/buffer exchange following tagmentation into Releasing Buffer (step 19).
Why did my reaction fail?
Pipetting 10 - 30 times and vortexing every 10 minutes in the Tagmentated DNA Releasing step 22 is essential. The experiment will not work without this.
Can all the reagents and kit components, including master mixes and enzymes, thaw out and be prepared at room temperature?
Everything except the Assembled Transposomes and Q5 DNA polymerase can be thawed at room temperature. The Assembled Transposomes and Q5 DNA Polymerase are formulated with glycerol and will not need thawing, just put these on ice. The other components can be frozen again and thawed again for future reactions, so not all 16 reactions need to be done simultaneously.
What is the expected final library size, and what should the library traces look like on a fragment analyzer?
Library fragments should range from around 250 bp to 1000 bp in length with an oscillation period ~150 bp. To see example traces, please visit the ATAC-Seq kit webpage.
How many sequencing reads are needed per sample?
Typically 30 million paired-end reads is sufficient, with 20 million being a minimum. The number of reads is dependent both on the sample genome and the end goal of the ATAC-Seq assay. If the sample genome is very large, or if more advanced analysis is required, more sequencing reads may be needed.
Can I multiplex more than 16 samples using the ATAC-Seq Kit?
The Fixed Cell ATAC-Seq Kit is supplied with 4x4 unique dual indexes for 16 unique samples. The indexed primers in the kit are identical to the Illumina Nextera primers corresponding to N701-N704 and N501-N504. If you would like to multiplex more than 16 samples our Nextera™-Compatible Multiplex Primers (96 plex) kit (Cat. No. 53155) enables multiplexing up to 96 reactions. These primers are provided at a concentration of 25 µM to be used directly in our Kits. You could also purchase and combine other Illumina Nextera primers at the same concentration (25 µM) as those in the kit.
How does the Fixed Cell ATAC-Seq kit protocol differ from the native ATAC-Seq kit?
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Fixed Cell ATAC-Seq Kit Documents
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| Name | Format | Cat No. | Price | |
|---|---|---|---|---|
| Fixed Cell ATAC-Seq Kit | 16 rxns | 53151 | ¥15,600 | Buy |
