Optimization of ATAC-Seq Protocols for Adherent Cells

Abstract

Optimization of ATAC-seq protocols for Adherent Cells

Introduction: Previous studies have shown that tissue mechanics (e.g., varying matrix stiffness) can enhance human embryonic stem cell lineage commitment into mesoderm lineages, however, the contribution of chromatin reorganization in this stiffness-mediated enhancement of lineage commitment has not been explored. Thus, we aim to provide a platform to measure changes in chromatin accessibility within cells adhered to matrices of variable stiffness. To do so, we hope to utilize Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq) protocols, however, there is a lack of literature utilizing ATAC-seq for adherent cells in order to see the open chromatin landscape.

Objective: Our Goal is to optimize ATAC-seq protocols using adherent cells through pre-assay formaldehyde fixation. We hypothesize that ATAC-seq protocols using fixed cells should yield similar results to normal assay conditions.

Design: For this experiment, we plated human embryonic stem cells (HuES64) to tissue culture plastic dishes. We preformed the ATAC-sec protocol on 50,000 stem cells. Stem cells were maintained in growth medium for 3 days until they reached up to 90% confluency. The primary methods used were ATAC-seq, SPRI bead purification, PCR, qPCR, and TapeStation analysis

Results: Our results section is based off of qPCR and TapeStation. We had 6 samples; 3 were washed, 2 were fixed and 1 was washed-unfixed. For qPCR, the results tell us how many more cycles needed to be ran in PCR. Our range was 6-10 extra PCR cycles to ensure we fully amplified our DNA samples. For TapeStation results, it told us how many base pairs we had in each six samples. Our base pair range was 600 to 100 base pairs per sample.

Conclusion: In conclusion, we were able to show that ATAC-seq can function with fixed cells. After successfully performing TapeStation analysis, samples will be sent for sequencing. Sequencing will determine if there are any possible differences in ATAC-seq output between fixed and normal cells due to transposase accessibility to fixed chromatin or if there is truly no bias from the fixation process. Fixation of cells worked well thus far with the ATAC-seq protocol, but if it can be performed directly on adherent cells is yet to be determined.

Future Work: Perform fixed ATAC-seq protocol on fixed adherent cells and run qPCR and TapeStation analysis to verify success of protocol. Perform fixed ATAC-Seq protocol on cells grown on different matrix stiffnesses. Process sequencing data using computational tools and identify regions with different chromatin landscape.