1. Single Cell Regulatory Variation
Clayton Barham

2. Goals of Studying Single Cell Regulatory Variation
Discover the mechanisms that cause different cells to have different chromatin structures and different expressions of regulatory elements
Determine how those differences affect cell phenotype and cell behavior

3. Applications
Better understand how stem cells differentiate and change their DNA regulation to adapt to their new specialization
Better understand the regulatory diversity within tumors, for more effective diagnosis and treatment
Better understanding how individual cells adapt their transcriptome in the face of exposure to chemicals may help better understand and counteract drug resistance

4. First Paper
Single-cell chromatin accessibility reveals principles of regulatory variation
Published in Nature, 2015, by Buenrostro et al.
Hypothesis: phenotypic diversity between individual cells stems from differences in chromatin accessibility between cells
Goal: develop a method, single cell ATAC-seq (scATAC-seq), to map the chromatin regions that are accessible in individual cells, which can be used to investigate specific problems

5. Background: ATAC-seq
scATAC-seq adapts ATAC-seq for use on individual cells
ATAC-seq is an ensemble method, exposing populations of cells to Tn5 transposase to tag exposed regions of chromatin

6. scATAC-seq Assay cells using Fluidigm microfluidics environment
Use PCR to amplify collected libraries
Pool and sequence single cell libraries

7. Measuring Regulatory Variation

8. Experimental Validation of scATAC-seq
Measure variability of multiple features between cells
Inhibit proteins associated with significant variability of specific features and observe results on scATAC-seq data
Measure sets of peaks that co-vary within individual cells, to reveal long range dependency in regulation

9. Results: Variability of Features Between Cells

10. Results: Inhibition of Factors Associated with Variability

11. Results: Co-dependent Peaks

12. Second Paper
Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation
Published in Cell, 2018, by Buenrostro et al.
Measure regulatory variation in differentiating adult stem cells

13. Background: Hematopoiesis
Human hematopoiesis involves differentiation of pluripotent adult stem cells
Process defined by Transcription Factors and their influence on chromatin

14. scATAC-seq Pipeline to Study Stem Cells

15. Experiments
Used scATAC-seq to analyze 10 populations of adult stem cells from blood and bone marrow
Combine scATAC-seq and RNA-seq to analyze the epigenome and transcriptome of Granulocyte-Macrophage Progenitor (GMP) cells
Integrate scATAC-seq and RNA-seq data to study how changes in regulatory elements affects chromatin accessibility and gene expression

16. Results: Regulatory Variation in 10 Adult Stem Cell Populations

17. Results: Transcriptome of GMP Cells

18. Results: Regulatory Elements and Motif Expression

19. References
J. D. Buenrostro et al., “Single-cell chromatin accessibility reveals principles of regulatory variation,” Nature, vol 523, pp. 486–490, July 23, 2015
J. D. Buenrostro et al., “Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation,” vol , pp E16, May 31, 2018