Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells - PubMed (original) (raw)

Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells

Pamela Milani et al. Sci Rep. 2016.

Abstract

In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it is difficult to perform this technique on frozen samples because freezing cells before extracting nuclei can impair nuclear integrity and alter chromatin structure, especially in fragile cells such as neurons. Our aim was to develop a protocol for freezing neuronal cells that is compatible with ATAC-Seq; we focused on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells (iMNs) from a patient affected by spinal muscular atrophy. We found that while flash-frozen iMNs are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved cells. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved iMNs quantitatively agree.

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Figures

Figure 1

Figure 1. Outline of ATAC-Seq procedure using fresh, flash-frozen, and cryopreserved iPSC-derived motor neurons.

The key experimental steps are nuclei extraction, transposase reaction, size selection, PCR amplification and sequencing. The quality control (QC) checkpoints consist of morphological evaluation of nuclei, agarose gel electrophoresis of libraries, and real-time qPCR to assess the enrichment of open-chromatin sites. (F = fresh, FF = flash-frozen, C = cryopreserved).

Figure 2

Figure 2. Fibroblast-derived iPSCs differentiate into SMI32- and ISL1-positive motor neurons.

Differentiated cells were labeled to evaluate the immunoreactivity of SMI32 (green) and ISL1 (red) proteins, two markers of mature motor neurons. Nuclei were stained with DAPI. Motor neurons were imaged with 10x magnification. The image on the right represents a higher magnification of selected neurons. Scale bar = 75 μm.

Figure 3

Figure 3. Representative results for ATAC-Seq carried out on fresh and flash-frozen cells.

(A) Nuclear morphological evaluation: nuclei from fresh cells were of high quality, while excessive clumping was observed for nuclei from flash-frozen neurons. (B) Agarose gel electrophoresis of libraries: the nucleosome phasing pattern on the gel was not detected in flash-frozen samples, as opposed to fresh cells. (C) ATAC-Seq tracks were visualized with the Gviz package: while we detected sharp peaks for fresh samples, the reads from flash-frozen neurons were distributed noisily across the genome. (F = fresh, FF = flash-frozen).

Figure 4

Figure 4. Representative results for ATAC-Seq carried out on fresh and cryopreserved cells.

(A) Nuclear morphological evaluation: similar to nuclei from fresh cells, nuclei from cryopreserved neurons were intact and of high quality. (B) Agarose gel electrophoresis of libraries: the nucleosome pattern on the gel was evident for both fresh and cryopreserved samples. (C) ATAC-Seq tracks were visualized with the Gviz package: peaks from both fresh and cryopreserved neurons were sharp and overlapped with H3K4me3 ChIP-Seq peaks from ENCODE (F = fresh, C = cryopreserved).

Figure 5

Figure 5. Real-time qPCR for the assessment of the quality of ATAC-Seq libraries.

(A) Genomic locations of the primers used to amplify positive (human GAPDH gene promoter) and negative (human gene desert region) control sites. (B) Fold enrichment of the open-chromatin site over the Tn5-insensitive site: while real-time qPCR experiments showed high enrichment for fresh and cryopreserved samples, poor results were obtained with flash-frozen cells. (F = fresh, FF = flash-frozen, C = cryopreserved).

Figure 6

Figure 6. Quantitative comparison of fresh and cryopreserved cells.

(A) Correlative analysis of the number of reads in 5 kb regions of the genome. The lower left triangle of the figure shows the scatter plots of the log2 read counts for each pair of technical replicates (5 kb regions with less than 10 read counts were excluded from the analysis). The upper right triangle displays the corresponding values of the Pearson correlation coefficient. (B) Average read profiles across the transcriptional start sites (TSS) using a 2.5 Kb window size. The overall pattern is very similar between fresh and cryopreserved iMNs. (C) Location-based distribution analysis: the distribution of neighboring genomic features to open-chromatin regions is highly similar between fresh and cryopreserved samples. (F = fresh, FF = flash-frozen, C = cryopreserved).

Figure 7

Figure 7. Differentially enriched sites detected between fresh and cryopreserved samples.

(A) The fold-change values for differentially enriched sites between fresh and cryopreserved samples are plotted as a function of the position of the sites across all genome. The changes were small (<3-fold). (B) Genomic tracks of ATAC-Seq results showing a differentially enriched site between fresh and cryopreserved samples. (C) Pie chart showing the genomic location distribution of the differentially enriched sites.

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