Microfluidics is a versatile tool with many applications in biology. Its ability to manipulate small volumes of liquid precisely has led to the development of many microfluidic assay platforms. They could handle small amounts of samples and carry out analysis with high sensitivity and throughput. Microfluidic assays have provided new insights into scarce biological samples at higher resolution. In this thesis, we developed microfluidic tools to conduct low input ChIP-seq and ChIRP-seq. We applied them to a variety of samples profiling different targets. The native MOWChIP-seq platform was developed to map RNA polymerase II, transcription factors and histone deacetylase binding in 1,000-50,000 cells. We examined mouse prefrontal cortex and cerebellum using this technology. We found extensive differences that correlated with distinct neurological functions of the brain regions. The same platform and workflow were used to profile five key histone modifications in human lung tumor and normal tissue samples. Integrative analysis with gene expression data revealed extensive chromatin remodeling in lung tumor. Spatial histone modification mapping was conducted in mouse neocortex in a similar fashion. We generated an epigenomic tomography that demonstrated the molecular state of the brain in 3D. Lastly, we developed a microfluidic version of the ChIRP-seq process which successfully conducted the assay using only 500K cells. This improvement makes ChIRP-seq in tissue samples feasible. / Doctor of Philosophy / Microfluidics is a type of technology that can control small volumes of liquid in a miniature system. It can carry out reactions on very small scales with higher precision and sensitivity than conventional methods. Microfluidics has found many uses in the field of biology, especially dealing with samples available in limited quantities. These low input microfluidic platforms have helped researchers gain new knowledge on many complex questions. In this thesis, we developed microfluidic tools to carry out low input ChIP-seq and ChIRP-seq. These are two established techniques used to map where certain targets are located on the genome of an organism. These targets include specific chemical modifications to the wrapper protein of DNA (histone modification), proteins that take part in transcription and expression of genes (RNA polymerase II, transcription factors) and other molecules. Our nMOWChIP-seq system removed the need for fixation by chemicals. It was able to examine RNA polymerase II, transcription factors and other enzymes using 1,000-50,000 cells. Traditional ChIP-seq requires more than 10 million cells and time-consuming chemical treatment steps. Our technology greatly improved sensitivity and ease of use. We also used this platform to test five important histone modifications in human lung tumors and healthy tissues. We constructed a spatial map of histone modification in mouse brain by analyzing slices of the cortex. Finally, we developed a microfluidic version of ChIRP-seq process to map locations of long non-coding RNAs in cultured human cells. The cells needed for a successful test were reduced to 500K from 20 million of the original workflow.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/116243 |
| Date | 07 September 2023 |
| Creators | Liu, Zhengzhi |
| Contributors | Department of Biomedical Engineering and Mechanics, Lu, Chang, Li, Liwu, Verbridge, Scott, Chen, Zhen Bouman, Davalos, Rafael V. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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