Highly scalable generation of DNA methylation profiles in single cells
Ontology highlight
ABSTRACT: We present a novel method: single-cell combinatorial indexing for methylation analysis (sci-MET), which is the first highly scalable assay for whole genome methylation profiling of single cells. We use sci-MET to produce 3,282 total single-cell bisulfite sequencing libraries and achieve read alignment rates of 68± 8%, comparable to those of bulk cell methods. As a proof of concept, we applied sci-MET to deconvolve the cellular identity of a mixture of three human cell lines. Next, we applied sci-MET to mouse cortical tissue, which successfully identified excitatory and inhibitory neuronal populations as well as non-neuronal cell types.
Project description:We developed a combinatorial indexing strategy to profile the transcriptomes of large numbers of single cells or nuclei (Single cell Combinatorial Indexing RNA-seq or sci-RNA-seq). We applied sci-RNA-seq to profile nearly 50,000 cells from C. elegans at the L2 stage, effectively ~56-fold “shotgun cellular coverage” of its somatic cell composition.
Project description:Single-cell combinatorial indexing (sci-) methods have addressed major limitations of throughput and cost for many single cell modalities. With the incorporation of linear amplification and 3-level barcoding in our suite of methods called sci-L3, we further addressed the limitations of uniformity in single cell genome amplification. Here, we build on the generalizability of sci-L3 by extending it to template strand sequencing (sci-L3-Strand-seq), genome conformation capture (sci-L3-Hi-C), and the joint profiling of RNA and chromatin accessibility (sci-L3-RNA/ATAC). We demonstrate the ease of adapting sci-L3 to these new modalities by only requiring a single-step modification of the original protocol. As a proof-of-principle, we show our ability to detect sister chromatid exchanges, genome compartmentalization, and cell state specific features in thousands of single cells. We anticipate sci-L3 to be compatible with additional modalities, including DNA methylation (sci-MET) and chromatin associated factors (CUT&Tag), and ultimately enable a multi-omics readout of them.
Project description:DNA accessibility of cis regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. To obtain a more comprehensive view of CRE dynamics, we applied single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq) to whole 24hpf stage zebrafish embryos thereby measuring DNA accessibility in ~23,000 single cells. We developed two solutions to computational challenges in analyzing single-cell accessibility maps: 1) selection of informative genome segments, and 2) genome-wide classification of both cell-type-specific and multi-cell-type accessibility dynamics. We validated the sci-ATAC-seq results with bulk measurements for histone post-translational modifications and 3D genome organization, recovering known relationships between chromatin modalities and providing additional regulatory classifications. Furthermore, we applied sci-ATAC-seq to cloche/npas4l mutant embryos which revealed known and novel cellular roles for the hemato-vascular transcriptional master regulator, and suggested an intricate network regulating its expression. These data and their extensive analysis constitute a valuable developmental, molecular, and computational resource for future studies.
Project description:A scalable, cost-effective method that combines CRISPR perturbations with a single-cell indexing assay for transposase-accessible chromatin (CRISPR-sciATAC). This method links genome-wide chromatin accessibility to genetic perturbations through simultaneous capture of ATAC-seq fragments and CRISPR guide RNAs from single cells using a 96-well plate combinatorial indexing approach.
Project description:We report a high depth single-cell combinatorial indexing (sci-)ATAC-seq map of the murine hippocampus from fresh and frozen tissue as well as cultured neurons
Project description:Single-cell RNA-seq libraries were generated using two and three level single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) of untreated or small molecule inhibitor exposed HEK293T, NIH3T3, A549, MCF7 and K562 cells. Different cells and different treatment were hashed and pooled prior to sci-RNA-seq using a nuclear barcoding strategy. This nuclear barcoding strategy relies on fixation of barcode containing well-specific oligos that are specific to a given cell type, replicate or treatment condition.
Project description:We devised an improved assay for single cell profiling of chromatin accessibility with three-level combinatorial indexing (sci-ATAC-seq3). We applied this method to 53 fetal tissue samples representing 15 organs, altogether profiling approximately one million single cells. We leveraged cell types defined by gene expression in the same organs to annotate these data, and built a catalog of hundreds-of-thousands of candidate gene regulatory elements exhibiting cell type-specific accessibility. Our analyses focus on the properties of lineage-specific transcription factors, organ-specific specializations of broadly distributed cell types, and cell type-specific enrichments of complex trait heritability. Additional data formats are available at atlas.brotmanbaty.org.
Project description:Gene expression is a dynamic process on multiple scales, e.g. the cell cycle, response to stimuli, normal differentiation and development, etc. However, nearly all techniques for profiling gene expression in single cells fail to directly capture these temporal dynamics, which limits the scope of biology that can be effectively investigated. Towards addressing this, we developed sci-fate, a new technique that combines S4U labeling of newly synthesized mRNA with single cell combinatorial indexing (sci-), in order to concurrently profile the whole and newly synthesized transcriptome in each of many single cells. As a proof-of-concept, we applied sci-fate to a model system of cortisol response, and characterized expression dynamics in over 6,000 single cells. From these data, we quantify the dynamics of the cell cycle and of glucocorticoid receptor activation, while also exploring their intersection. We furthermore use these data to develop a framework for estimating cell state transition probabilities, and to identify factors whose dynamic expression potentially regulates these transitions. The experimental and computational methods described here may be broadly applicable to quantitatively characterize cell state dynamics in in vitro systems.
Project description:Here we describe sci-CAR, a combinatorial indexing strategy to jointly profile chromatin accessibility and mRNA in each of thousands of single cells. As a proof-of-concept, we apply sci-CAR to 4,825 cells comprising a time-series of dexamethasone treatment, as well as to 11,233 cells from the mouse kidney.
Project description:Single cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here we report a simplified, optimized version of the three-level sci-RNA-seq protocol that is faster, higher yield, more robust, and more sensitive, than the original sci-RNA-seq3 protocol, with reagent costs on the order of 1 cent per cell or less. We showcase the optimized protocol via whole organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a “tiny sci-*” protocol for experiments where input is extremely limited.