Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits

Abstract

Skeletal muscle accounts for the largest proportion of human body mass, on average, and is a key tissue in complex diseases and mobility. It is composed of several different cell and muscle fiber types. Here, we optimize single-nucleus ATAC-seq (snATAC-seq) to map skeletal muscle cell–specific chromatin accessibility landscapes in frozen human and rat samples, and single-nucleus RNA-seq (snRNA-seq) to map cell-specific transcriptomes in human. We additionally perform multi-omics profiling (gene expression and chromatin accessibility) on human and rat muscle samples. We capture type I and type II muscle fiber signatures, which are generally missed by existing single-cell RNA-seq methods. We perform cross-modality and cross-species integrative analyses on 33,862 nuclei and identify seven cell types ranging in abundance from 59.6% to 1.0% of all nuclei. We introduce a regression-based approach to infer cell types by comparing transcription start site–distal ATAC-seq peaks to reference enhancer maps and show consistency with RNA-based marker gene cell type assignments. We find heterogeneity in enrichment of genetic variants linked to complex phenotypes from the UK Biobank and diabetes genome-wide association studies in cell-specific ATAC-seq peaks, with the most striking enrichment patterns in muscle mesenchymal stem cells (∼3.5% of nuclei). Finally, we overlay these chromatin accessibility maps on GWAS data to nominate causal cell types, SNPs, transcription factor motifs, and target genes for type 2 diabetes signals. These chromatin accessibility profiles for human and rat skeletal muscle cell types are a useful resource for nominating causal GWAS SNPs and cell types.

Single-nuclei Transcriptome of Human AT Reveals Metabolically Distinct Depot-Specific Adipose Progenitor Subpopulations

“…Future steps will also include the addition of single nuclear ATAC strategies and multiomic analysis to identify the regulatory regions that are responsible for the gene expression changes as has been recently performed for skeletal muscle in diabetes. 58…”

Human skeletal muscle ageing atlas

“…To date, most studies have focused on one particular mechanism or cell type in the muscle, and lacked a comprehensive approach to fully understand muscle ageing. To address this, recent mouse and human skeletal muscle studies pioneered the use of either single-cell (scRNA-seq) [17][18][19][20][21] or single-nucleus RNA sequencing (snRNA-seq) [22][23][24][25][26] to understand muscle cell type heterogeneity as well as their changes in ageing. Both sc-and snRNA-seq approaches have their own limitations when applied to muscle: droplet single-cell sequencing approaches are not well suited for myofiber profiling due to the multinuclearity and large size of fibres, while single-nucleus sequencing often lacks resolution for the less-abundant MuSCs and microenvironment cell types.…”

A Protocol for Single Nucleus RNAseq from Frozen Skeletal Muscle

“…Increasingly, this technique is proving well suited to simultaneously examining many cell types, uncover rare disease states, and identify subpopulations with distinct functions (Birnbaum, 2018). Few studies have examined human skeletal muscle at the single cell level (Barruet et al, 2020;De Micheli, Spector, Elemento, & Cosgrove, 2020;Orchard et al, 2021;Rubenstein et al, 2020), however, its utility is clear for basic and medical science studies of muscle in health and disease.…”

“…For this reason, single cell data typically include very low proportions of myofibers (De Micheli et al, 2020). Conversely, isolating nuclei from muscle yields very high proportions of myonuclei (Dos Santos et al, 2020;Orchard et al, 2021;Petrany et al, 2020). Single nuclear RNA sequencing is the best option if the experimental objective is to obtain sequencing data from all cell types in a muscle biopsy.…”

“…Few papers have applied snRNAseq to skeletal muscle (Dos Santos et al, 2020;Jiang et al, 2020;Kim et al, 2020;Orchard et al, 2021;Petrany et al, 2020;Zeng et al, 2016). Of these, three groups isolated nuclei directly from mouse muscle (Dos Santos et al, 2020;Kim et al, 2020;Petrany et al, 2020), two used cell cultures (Jiang et al, 2020;Zeng et al, 2016), and only one used human skeletal muscle, although they focussed on analyzing accessible chromatin regions by means of ATAC-seq (Orchard et al, 2021). To isolate nuclei directly from muscle, these reports used fiber dissection and dounce homogenization (Dos Santos et al, 2020), or a mix of various homogenization methods and detergents (Orchard et al, 2021;Petrany et al, 2020).…”