An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000–33,000 accessible chromatin regions in lamina and 22–61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments.
Digital Object Identifier (DOI)
Funding was provided by the Grayson Jockey Club Foundation, USDA NRSP-8, and the UC Davis Center for Equine Health. Funding Support for CF was provided by the National Institutes of Health (NIH) (L40 TR001136). None of the funding agencies had any role in the design of the study, analysis, interpretation of the data, or writing of the manuscript. Salary support for SP was provided by the Ann T. Bowling Fellowship at the UC Davis Veterinary Genetics Laboratory.
ATAC-seq data used in this study are available from the European Nucleotide Archive under the accession PRJEB41317. RNA-seq data from the liver and lamina tissues of the same two animals used in this study can be found from the European Nucleotide Archive under the accession PRJEB26787.
Peng, Sichong; Bellone, Rebecca; Petersen, Jessica L.; Kalbfleisch, Ted S.; and Finno, Carrie J., "Successful ATAC-Seq from Snap-Frozen Equine Tissues" (2021). Veterinary Science Faculty Publications. 54.
Data sheet 1
Data_Sheet_2_Successful ATAC-Seq From Snap-Frozen Equine Tissues.docx (22 kB)
Data sheet 2
Image_1_Successful ATAC-Seq From Snap-Frozen Equine Tissues.TIF (1413 kB)
Supplementary Figure 1: Fragment size distributions of libraries from L1 as determined by sequencing and Fragment Analyzer.
Image_2_Successful ATAC-Seq From Snap-Frozen Equine Tissues.TIF (1408 kB)
Supplementary Figure 2: Fragment size distributions of libraries from L2 as determined by sequencing and tapestation.
Image_3_Successful ATAC-Seq From Snap-Frozen Equine Tissues.TIF (320 kB)
Supplementary Figure 3: Duplication and mitochondrial contamination rates. (A) Total, mitochondrial, and unique nuclear read counts of all libraries; (B) Comparison between first sequencing run (left) and combined reads (right) from L1 liver CN libraries; (C) Fingerprint plot of L1 CN liver libraries.
Image_4_Successful ATAC-Seq From Snap-Frozen Equine Tissues.TIF (366 kB)
Supplementary Figure 4: MACS2 peak calling statistics. (A) Number of peaks, (B) peak length distribution, (C) peak score distribution, and (D) percent of genome covered by peaks for each library. (E,F) Peak metrics assessed using ChIP-seq dataset in liver (E) and lamina (F) libraries.