When it comes to love, being single has its pros and cons. This is also true for cells, single cells give a lot high-resolution picture, but it’s often very difficult to get enough material. While other single cell methods have flourished with low inputs, studies of the heterogeneity of chromatin states by single cell chromatin immunoprecipitation sequencing (scChIP-seq) have remained challenging.
To address the single cell ChiP-seq woes, the labs of Andrew Griffiths, Céline Vallot, and Annabelle Gérard (Université PSL, Paris) developed a droplet microfluidics approach to profile the chromatin landscapes of thousands of cells at single-cell resolution. Their scChIP-seq approach can cover up to 10,000 loci per cell. Two sets of oppositely charged droplets are created and fused, the first containing a hydrogel bead carrying barcoding adapters an the other containing individual cells. After nucleosomes from single cells are uniquely barcoded, immunoprecipitation against the histone mark of interest and sequencing are performed. The barcodes allow for peaks of histone mark enrichment to be mapped back to individual cells. To test the technique, they examined H3K4me3 and H3K27me3 in cell lines and patient-derived xenograft models of acquired resistance to chemotherapy. Here’s what they found:
and T cell lines processed separately then pooled for IP show high correlation
between technical and biological replicates with no batch effect.
- The cell types were clearly discerned using t-SNE clustering
- scChIP-seq analysis of a patient-derived mouse
xenograft model with and without acquired resistance to capecitabine
(chemotherapy) revealed novel insights
- Stromal cells clustered into three distinct groups based on H3K27me3 profile regardless of chemotherapy sensitivity while scRNA-seq identified four groups
- The primary tumor cells clustered according to their chemotherapy resistance for both H3K27me3 and RNA-seq with the resistant population being more heterogeneous
- Assessment of H3K27me3 in a pair of
estrogen-receptor positive breast cancer cell lines sensitive and resistant to
tamoxifen identified a small (16%) group of cells within the sensitive tumor
shared chromatin features with all resistant cells
- The same proportion (17%) displayed expression of genes characteristic of resistant tumor cells
- This suggests that non-genetic features characteristic of resistant cells are already found in cells from the sensitive tumor
This new scCHIP-seq method represents a substantial advance in single cell chromatin profiling and single cell sequencing in general. Their droplet fusion approach allows for segmentation of cell populations based on their chromatin landscape and the identification of key chromatin features within each subpopulation. This proof of principle study probing cancer cell dynamics presents novel techniques that can be applied to other complex biological systems.
So, if you’re looking for your cells to be single and ready to mingle check out this new approach in Nature Genetics, June 2019.