Tobias Hohl (MPI): Sample handling in scATAC-seq and astrocytogenesis – two single-cell stories.

Abstract

Assay for transposase-accessible chromatin (ATAC) sequencing (seq) revolutionized the field of
epigenetics since its emergence by providing a means to uncover chromatin dynamics and
other factors affecting gene expression. The development of single-cell (sc) applications in
recent years led to an even deeper understanding of cell type specific gene regulatory
mechanisms. One of the major challenges while running ATAC-seq experiments, bulk or sc, is
the need for good cell viability for successful experiments. While various freezing methods have
already been tested and established for bulk and sc ATAC-seq, quality metrics for flash frozen
or cryopreserved samples are rather poor when compared to fresh samples. This makes it
difficult to conduct all sorts of complex experiments i.e. with multiple conditions, patients, or
time course studies. Especially, accounting for batch effects can be difficult if samples need to
be processed on different days. We tackled this issue by combining fixation protocols with
freezing methods for both bulk and sc ATAC-seq library preparation protocols and show that an
additional fixation step can improve library quality and yield data comparable to fresh samples.
Additionally, we set up a sample multiplexing protocol for scATAC-seq experiments to cut down
on costs for library preparation and are working on optimizing the protocol.

On a different project, we generated a comprehensive single-cell multi-omics dataset with
temporal resolution to investigate neuronal development. Astrocytes, a subtype of glial cells
making up the majority of cells in a mature central nervous system (CNS), serve a huge variety
of functions in the mammalian brain. Canonically, astrocytes derive from Emx1-positive
pluripotent progenitors that switch from cortical neurogenesis to astrocytogenesis in later
development. Our data suggests the presence of an alternative, Emx1-independent,
fate-restricted pathway producing astrocytes in parallel to cortical neurogenesis. These results
provide novel insights into the cellular mechanisms of astrocytogenesis and hint the presence of
an additional fate-restricted progenitor subtype.