Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay.

Chromatin accessibility captures in vivo protein-chromosome binding status, and is considered an informative proxy for protein-DNA interactions. DNase I and Tn5 transposase assays require thousands to millions of fresh cells for comprehensive chromatin mapping. Applying Tn5 tagmentation to hundreds of cells results in sparse chromatin maps. We present a transposome hypersensitive sites sequencing assay for highly sensitive characterization of chromatin accessibility. Linear amplification of accessible DNA ends with in vitro transcription, coupled with an engineered Tn5 super-mutant, demonstrates improved sensitivity on limited input materials, and accessibility of small regions near distal enhancers, compared with ATAC-seq

The Single Cell Transposome Hypersensitive Sites Sequencing (scTHS-seq) assay for Chromatin Accessibility and Assessment of Epigenetic States in the Human Adult Brain

Chromatin accessibility captures in vivo protein-chromosome binding status, and is considered an informative proxy for protein-DNA interactions. DNase I and Tn5 transposase assays require thousands to millions of fresh cells for comprehensive chromatin mapping. Applying Tn5 tagmentation to hundreds of cells and to single cells results in sparse chromatin maps at high throughput or dense chromatin maps at low throughput. We present a high throughput transposome hypersensitive sites sequencing assay for highly sensitive characterization of chromatin accessibility. Linear amplification of accessible DNA ends with in vitro transcription, coupled with an engineered Tn5 super-mutant, demonstrates improved sensitivity on limited input materials, and accessibility of small regions near distal enhancers, compared with ATAC-seq. Application to single cells was achieved by adapting THS-seq to combinatorial indexing to generate high sensitivity DNA accessibility maps for tens of thousands of single cells from the human adult visual and frontal cortex. Integrative analysis of snDrop-seq and scTHS-seq has allowed us to identify transcription factors and regulatory elements shaping the state of different brain cell types, and to map genetic risk factors of common human brain diseases to specific pathogenic cell types and subtypes. Overall we demonstrate the viability of scTHS-seq to quantitate DNA accessibility in single cells, and application to biological samples to shed insight on human brain diseases

A single-cell regulatory map of postnatal lung alveologenesis in humans and mice.

Ex-utero regulation of the lungs' responses to breathing air and continued alveolar development shape adult respiratory health. Applying single-cell transposome hypersensitive site sequencing (scTHS-seq) to over 80,000 cells, we assembled the first regulatory atlas of postnatal human and mouse lung alveolar development. We defined regulatory modules and elucidated new mechanistic insights directing alveolar septation, including alveolar type 1 and myofibroblast cell signaling and differentiation, and a unique human matrix fibroblast population. Incorporating GWAS, we mapped lung function causal variants to myofibroblasts and identified a pathogenic regulatory unit linked to lineage marker FGF18, demonstrating the utility of chromatin accessibility data to uncover disease mechanism targets. Our regulatory map and analysis model provide valuable new resources to investigate age-dependent and species-specific control of critical developmental processes. Furthermore, these resources complement existing atlas efforts to advance our understanding of lung health and disease across the human lifespan

Additional file 1: Figure S1. of Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay

Schematic of transposon design and transposome complex generation. Figure S2. Validation of successful transposon insertion and DNA fragmentation. Figure S3. Validation of T7 in vitro transcription. Figure S4. Validation of PCR amplification for barcode addition. Figure S5. Tn5059 concentration and dimethylformamide titrations for optimal in vitro transcription amplification yields. Figure S6. Comparison of peak region distances to transcription start sites. Figure S7. Significant gene ontology biological processes categories for each peaks dataset. Figure S8. Quantitation of mitochondrial reads in all datasets. Figure S9. 100-cell THS-seq/Tn5059 read statistics analysis. Figure S10. Peak overlap comparisons in all datasets. Figure S11. Base pair overlap comparisons in all datasets. Figure S12. Examination of peak overlap in a pairwise comparison of all experimental datasets. Figure S13. Examination of base pair overlap in a pairwise comparison of all experimental datasets. Figure S14. Two hundred kilobyte view of accessible chromatin marks in 500-cell datasets. Figure S15. Overview of chromatin accessibility at gene loci enriched in 100- and 500-cell THS-seq/Tn5059 data and not enriched in 500-cell ATAC-seq/EzTn5 data. Figure S16. Overview of chromatin accessibility at major genes implicated in cancer and in immune system function. Figure S17. Peak size distribution comparisons between THS-seq, ATAC-seq, and ENCODE data. Figure S18. Validation of peaks based on peak length. Figure S19. THS-seq and ATAC-seq peak capture preferences. Figure S20. Comparison of 500-cell ATAC-seq/EzTn5 data to published ATAC-seq data. Figure S21. Two hundred kilobyte view of accessible chromatin in ATAC-seq/EzTn5 data versus published ATAC-seq data. Table S1. 100-cell THS-seq/Tn5059 data and controls with read statistics and data analysis. Table S2. Peak size counts versus lengths of Dfilter called peaks for each sample. Table S3. Analysis of datasets down-sampled to 8,351,125 unique reads and analysis of published ATAC-seq datasets. Table S4. Original datasets analysis results. Table S5. THS-seq oligo sequences

Liver-Derived IGF-I Contributes to GH-Dependent Increases in Lean Mass and Bone Mineral Density in Mice with Comparable Levels of Circulating GH

The relative contributions of circulating and locally produced IGF-I in growth remain controversial. The majority of circulating IGF-I is produced by the liver, and numerous mouse models have been developed to study the endocrine actions of IGF-I. A common drawback to these models is that the elimination of circulating IGF-I disrupts a negative feedback pathway, resulting in unregulated GH secretion. We generated a mouse with near total abrogation of circulating IGF-I by disrupting the GH signaling mediator, Janus kinase (JAK)2, in hepatocytes. We then crossed these mice, termed JAK2L, to GH-deficient little mice (Lit). Compound mutant (Lit-JAK2L) and control (Lit-Con) mice were treated with equal amounts of GH such that the only difference between the two groups was hepatic GH signaling. Both groups gained weight in response to GH but there was a reduction in the final weight of GH-treated Lit-JAK2L vs. Lit-Con mice. Similarly, lean mass increased in both groups, but there was a reduction in the final lean mass of Lit-JAK2L vs. Lit-Con mice. There was an equivalent increase in skeletal length in response to GH in Lit-Con and Lit-JAK2L mice. There was an increase in bone mineral density (BMD) in both groups, but Lit-JAK2L had lower BMD than Lit-Con mice. In addition, GH-mediated increases in spleen and kidney mass were absent in Lit-JAK2L mice. Taken together, hepatic GH-dependent production of IGF-I had a significant and nonredundant role in GH-mediated acquisition of lean mass, BMD, spleen mass, and kidney mass; however, skeletal length was dependent upon or compensated for by locally produced IGF-I

Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain.

Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, as well as computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for the study of complex processes in the brain, such as genetic programs that coordinate adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, which provided insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues

Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain

Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, and computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus Droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive-site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from the human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for studying complex processes in the brain, such as genetic programs coordinating adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, providing insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues

Single nucleus analysis of the chromatin landscape in mouse forebrain development

ABSTRACT Genome-wide analysis of chromatin accessibility in primary tissues has uncovered millions of candidate regulatory sequences in the human and mouse genomes 1–4 . However, the heterogeneity of biological samples used in previous studies has prevented a precise understanding of the dynamic chromatin landscape in specific cell types. Here, we show that analysis of the transposase-accessible-chromatin in single nuclei isolated from frozen tissue samples can resolve cellular heterogeneity and delineate transcriptional regulatory sequences in the constituent cell types. Our strategy is based on a combinatorial barcoding assisted single cell assay for transposase-accessible chromatin 5 and is optimized for nuclei from flash-frozen primary tissue samples (snATAC-seq). We used this method to examine the mouse forebrain at seven development stages and in adults. From snATAC-seq profiles of more than 15,000 high quality nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell-types in foetal and adult forebrains. We further define cell-type specific cis regulatory sequences and infer potential master transcriptional regulators of each cell population. Our results demonstrate the feasibility of a general approach for identifying cell-type-specific cis regulatory sequences in heterogeneous tissue samples, and provide a rich resource for understanding forebrain development in mammals

Author Correction: Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

In the version of this article initially published online, the accession code was given as GSE1000333. The correct code is GSE100033. The error has been corrected in the print, HTML and PDF versions of the article