Integrative genomic analysis of the human response to glucocorticoids

National Institute Of Allergy And Infectious Diseases

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Abstract

This was the first year of the project. We began by setting up the laboratory and tissue culture areas, validating and developing assays and protocols for tissue culture, nucleic acid purification, nucleic acid quality control, flow cytometry, and next-generation sequencing. We analyzed data from an initial experiment comparing the transcriptional response to glucocorticoids in a hematopoietic (human lymphoblastoid cells) and a non-hematopoietic (human fibroblasts) cell line. We generated an initial list of genes and long non-coding RNAs that responded differently to the glucocorticoid stimulus in the immune cell type when compared to the non-immune cell type. We then began work on primary human hematopoietic cells. Five different hematopoietic cell sub-populations were isolated from a healthy human donor. The cells were exposed in short-term culture to a glucocorticoid stimulus and RNA samples were obtained at multiple time points before and after the stimulus. A parallel experiment was performed on a non-hematopoietic cell type. We performed whole-RNA sequencing and small-RNA sequencing on the samples and are now analyzing the data. We are also working actively on optimizing the culture conditions for four additional non-hematopoietic primary cells, with the goal of performing similar time-series experiments