Reveal reviewer identity to authors (required): Yes

General assessment and major comments (Required): Spektor and colleagues have carried out single cell ATAC-seq to map the open chromatin landscape throughout the genome in relation to Down Syndrome, the most common genetic disorder linked to cognitive deficits. To do this, the authors use a well-known mouse model of Down Syndrome (Ts65Dn) and compare cellular and neuronal composition of two control and two Down Syndrome mice. The biggest change is predicted in cell-lineage for cortical interneurons (inhibitory) with an increase of up to 50% of interneurons in Ts65Dn cortices. The work is certain to be of great importance as it provides the first single cell resource (to my knowledge) of cell atlas identity for the Down Syndrome community working on corticogenesis. It will also be of interest to the wider neuroscience community. The paper is very well written and the data set seem of high quality and reproducibility.

However, I am not always convinced by the novelty of the biological findings (the imbalance between excitatory and inhibitory neurotransmission is known in Down Syndrome). My main concern relates to the lack of functional assays to wrap up the study. I was intrigued to see an abundance of dopamine medium spiny neurons accessible interneurons in the cortex but no statistical significant change in parvalbumin and somatostatin accessible interneurons. Could the authors include experimental validation work to support these findings, for example using immunohistochemistry or immunofluorescence, to quantify experimentally the densities of these neuronal populations in the Ts65Dn mouse adult cortex.

Minor Comments: Additional comments

Biological replicates: From the result section, I understand that two littermate control (2n) and two Ts65Dn (Ts) mice were used for the study. Can the authors add the number of assessed biological replicates in the Methods section including animal age, gender and mouse strain genetic background?

Cortices preparation: Considering the neuroanatomical defects reported in Down Syndrome patients in particular in entorhinal cortex and prefrontal cortex, could the authors be more specific in the text about the cortical regions dissected. Was the cortex as a whole processed (i.e. motor, somatosensory, piriform, cingulate, retrosplenial, entorhinal, visual) or was a selection of regions made?

Library preparation: Do the authors account for the hypo myelination and increased cell death observed in Down Syndrome brain samples. Related to this question, were the number of nuclei eliminated in Ts mice higher?

Gender selection: Is there an excess of male patients in Down Syndrome that would explain why male samples were processed and not females ? It would be useful to add this justification in the manuscript.

Additional data files and statistical comments: Statistics: Can the authors comment on the sensitivity of cell type detection relative to previous studies and what is the fold increase in read depth per nucleus compared to previous reports? It will be useful to have these information in the methods section.