A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex

Abstract

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1–3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas—containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities—is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

A multimodal cell census and atlas of the mammalian primary motor cortex

“…3b). We co-clustered L5 ET cells from the Epi-retro-seq data and the snRNA-seq 10x v3 B data 37 , and found that the consensus transcriptomic cluster L5 ET_1 corresponded to Epi-retro-seq clusters 0, 2 and 3, whereas transcriptomic clusters L5 ET_2-4 corresponded to Epi-retro-seq clusters 1, 4 and 5, which contain almost no MY-projecting neurons (Fig. 8d).…”

“…We describe a synthesis of eleven companion studies through a coordinated multi-laboratory effort. In these studies, we derive a cross-species consensus molecular taxonomy of cell types using scRNA-seq or single-nucleus RNA sequencing (snRNA-seq), DNA methylation and chromatin accessibility data [37][38][39][40] . In mouse, we map the spatial cellular organization by multiplexed error-robust fluorescence in situ hybridization (MERFISH) 41 , characterize morphological and electrophysiological properties by multimodal profiling using patch clamp recording, biocytin staining and scRNA-seq (Patch-seq) 42,43 , describe the cellular input-output wiring diagrams by anterograde and retrograde tracing 44 , identify glutamatergic neuron axon projection patterns by Epi-retro-seq 45 , Retro-MERFISH 41 and single-neuron complete morphology reconstruction 46 , and describe transgenic driver lines targeting glutamatergic cell types on the basis of marker genes and lineages 47 .…”

“…A mouse MOp molecular taxonomy was derived from seven scRNA-seq and snRNA-seq (sc/snRNA-seq) datasets and single-nucleus methylcytosine sequencing (snmC-seq2) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) datasets 37 . The combined sc/snRNA-seq datasets contained a large number of cells profiled using both droplet-based and deep full-length sequencing methods (Extended Data Table 1), resulting in a consensus transcriptomic taxonomy with the greatest resolution compared with other data types, including 90 neuronal and 116 total clusters or transcriptomic types (t-types) 37 . We used this mouse MOp transcriptomic taxonomy as the anchor for comparison and cross-correlation of cell-type classification results across all data types.…”

“…We used this mouse MOp transcriptomic taxonomy as the anchor for comparison and cross-correlation of cell-type classification results across all data types. We further applied two computational approaches, SingleCellFusion (SCF) and LIGER, to combine the transcriptomic and epigenomic datasets and derive an integrated molecular taxonomy consisting of 56 neuronal cell types (corresponding to the 90 transcriptomic neuronal types) 37 (Fig. 1a).…”

“…We patched more than 1,300 neurons in MOp of adult mice, recorded their electrophysiological responses to a set of current steps, filled them with biocytin to recover their morphologies (around 50% of cells) and obtained their transcriptomes using Smart-seq2 sequencing 42 . We mapped these cells to the mouse MOp transcriptomic taxonomy 37 (Fig. 1a).…”