Massively multiplex chemical transcriptomics at single-cell resolution.

High-throughput chemical screens typically use coarse assays such as cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here, we introduce "sci-Plex," which uses "nuclear hashing" to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof of concept, we applied sci-Plex to screen three cancer cell lines exposed to 188 compounds.

The single-cell transcriptional landscape of mammalian organogenesis.

Mammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution.

Joint profiling of chromatin accessibility and gene expression in thousands of single cells.

Although we can increasingly measure transcription, chromatin, methylation, and other aspects of molecular biology at single-cell resolution, most assays survey only one aspect of cellular biology. Here we describe sci-CAR, a combinatorial indexing-based coassay that jointly profiles chromatin accessibility and mRNA (CAR) in each of thousands of single cells. As a proof of concept, we apply sci-CAR to 4825 cells, including a time series of dexamethasone treatment, as well as to 11,296 cells from the adult mouse kidney.

A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility.

We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in ∼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and ∼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types.

The cis-regulatory dynamics of embryonic development at single-cell resolution.

Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear.

Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube.

Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here we demonstrate barcode partitioning of long DNA molecules in a single compartment using "on-bead" barcoded tagmentation.

Contiguity-Preserving Transposition Sequencing (CPT-Seq) for Genome-Wide Haplotyping, Assembly, and Single-Cell ATAC-Seq.

Most genomes to date have been sequenced without taking into account the diploid nature of the genome. However, the distribution of variants on each individual chromosome can (1) significantly impact gene regulation and protein function, (2) have important implications for analyses of population history and medical genetics, and (3) be of great value for accurate interpretation of medically relevant genetic variation. Here, we describe a comprehensive and detailed protocol for an ultra fast (<3 h library preparation), cost-effective, and scalable haplotyping method, named Contiguity Preserving Transposition sequencing or CPT-seq (Amini et al.

Sequencing thousands of single-cell genomes with combinatorial indexing.

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants.

PRODUCTION OF A PRELIMINARY QUALITY CONTROL PIPELINE FOR SINGLE NUCLEI RNA-SEQ AND ITS APPLICATION IN THE ANALYSIS OF CELL TYPE DIVERSITY OF POST-MORTEM HUMAN BRAIN NEOCORTEX.

Next generation sequencing of the RNA content of single cells or single nuclei (sc/nRNA-seq) has become a powerful approach to understand the cellular complexity and diversity of multicellular organisms and environmental ecosystems. However, the fact that the procedure begins with a relatively small amount of starting material, thereby pushing the limits of the laboratory procedures required, dictates that careful approaches for sample quality control (QC) are essential to reduce the impact of technical noise and sample bias in downstream analysis applications. Here we present a preliminary framework for sample level quality control that is based on the collection of a series of quantitative laboratory and data metrics that are used as features for the construction of QC classification models using random forest machine learning approaches.

Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing.

Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly.

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D.

In vitro, long-range sequence information for de novo genome assembly via transposase contiguity.

We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to > 1 megabase. These pools are "subhaploid," in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome.

Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing.

Haplotype-resolved genome sequencing enables the accurate interpretation of medically relevant genetic variation, deep inferences regarding population history and non-invasive prediction of fetal genomes. We describe an approach for genome-wide haplotyping based on contiguity-preserving transposition (CPT-seq) and combinatorial indexing. Tn5 transposition is used to modify DNA with adaptor and index sequences while preserving contiguity.