Project description:Joint profiling of chromatin accessibility and gene expression from the same single cell provides critical information about cell types in a tissue and cell states during a dynamic process. These emerging multi-omics techniques help the investigation of cell-type resolved gene regulatory mechanisms. Here, we developed in situ SHERRY after ATAC-seq (ISSAAC-seq), a highly sensitive and flexible single cell multi-omics method to interrogate chromatin accessibility and gene expression from the same single cell. We demonstrated that ISSAAC-seq is sensitive and provides high quality data with orders of magnitude more features than existing methods. Using the joint profiles from thousands of nuclei from the mouse cerebral cortex, we uncovered major and rare cell types together with their cell-type specific regulatory elements and expression profiles. Finally, we revealed distinct dynamics and relationships of transcription and chromatin accessibility during an oligodendrocyte maturation trajectory.
Project description:This study used droplet-based snATAC-seq to profile the chromatin accessibility landscape of 91,922 nuclei in the mouse cerebellum across eleven developmental stages, from the beginning of neurogenesis (e10.5) till adulthood (P63). The study included two biological replicates per stage, one from each sex. Cerebelli were dissected as whole or in two halves, nuclei were extracted and profiled using 10x single-cell ATAC reagent kit (v1.0) and a Chromium controller. Libraries were sequenced using paired-reads on Illumina NextSeq 550 and initial data processing was performed using Cellranger ATAC (1.1).
Project description:This study used droplet-based snATAC-seq to profile the chromatin accessibility landscape of 19,204 nuclei in the opossum (Monodelphis domestical) cerebellum across two developmental stages (postnatal day 21 and adult). The study included two biological replicates per stage, one from each sex, and an additional adult sample enriched for white matter. Cerebelli were dissected in two halves, nuclei were extracted from one half and profiled using 10x single-cell ATAC reagent kit (v1.1) and a Chromium controller. The white matter enriched sample was dissected from coronal cerebellum slices. Libraries were sequenced using paired-reads on Illumina NextSeq 550 and initial data processing was performed using Cellranger ATAC (1.1).
Project description:The aim of the experiment was to identify HAND1 target genes and its impact on chromatin accessibility in relation to cardiac development. A HAND1-null hESC line was used, in which a doxycycline-inducible HAND1-T2A-BFP transgene had been integrated in approximately half of the cells for HAND1 rescue / overexpression. The hESCs were differentiated with BMP4, Activin A and CHIR. On day 2.5, doxycycline was added. On day 3, cells were dissociated and sorted by BFP level using FACS. Samples were immediately processed for RNA-seq and ATAC-seq.
Project description:The experiment aimed to resolve cellular heterogeneity in cardiac differentiation through the purification of different cell populations by lineage markers and analysis of their transcriptomes and chromatin accessibility. The differentiation protocol was designed to promote cell diversity. The addition of SB at day 2, inhibited SMAD2/3 phosphorylation and created a high BMP signalling bias to restrict cardiac differentiation in favour of other mesodermal lineages, whereas the addition of DMH1 at day 2, inhibited SMAD1/5/8 phosphorylation to create a high Activin signaling bias to promote the co-differentiation of endoderm. Cells were sorted based on SOX17-tomato and NKX2-5-GFP knock-in reporters into their major classes: Pop1 = SB_G0_T0 Pop2 = SB_G1_T0 Pop3 = CTRL_G0_T0 Pop4 = CTRL_G1_T0 Pop5 = DM_G0_T0 Pop6 = DM_G1_T0 Pop7 = DM_G1_T1 Pop8 = DM_G0_T1
Project description:A map of open chromatin in control and Irx3-KO beige ME3 preadipocytes cells on days 0, 1 and 7 of adipogenic differentiation. Cells were differentiated for indicated number of days, pelleted and snap-frozen in liquid nitrogen. After thawing, cells were treated with DNase to remove exogenous DNA released from cells that died prior to freezing. Remaining cells were lysed in ATAC-seq lysis buffer from the ATAC-seq. Crude nuclear extracts was isolated by centrifugation and resuspended in Tagmentation buffer. Nuclear integrety was verified under microscope, and 50,000 nuclei were transferred for tagmentation. Tagmentation and indexing was performed according to the instructions of the ATAC-seq Kit (Active Motif Catalog No. 53150). Library QC was performed on a Bioanalyzer, and the library was sequenced on a HiSeq4000 Illumina platform using 75 bp paired end sequencing. Reads were filtered to remove low quality mappings, duplicates and mitochondrial mappings. Peaks were called by Macs2 and ENCODE blacklisted regions were excluded. Differentially open chromatin was identified by featurecounts and DESeq2.
Project description:Here, we generated multiome (coupled scRNA-seq and scATAC-seq) data over a time course of mid- and hindbrain organoid development to map cell composition and explore the regulatory mechanisms underlying cell type maturation. The dataset incorporates 5 time points from day 7 to day 120 from 3 human induced pluripotent stem cell (iPSC) lines using 2 previously established protocols. The protocols differentially generate ventral and dorsal cell types, and together cover regions such as floor-plate, dorsal and ventral midbrain, cerebellum, and additional parts of hindbrain. Comparing the data to a reference atlas of the developing human brain and to an integrated neural organoid cell atlas, we find that the gene regulatory architecture in organoid cells is predictive of primary counterparts and also that multiple regions are under-represented in existing organoid models.
Project description:Multi-modal profiling of different molecular layers from the same single cell enables more comprehensive characterisations of cellular heterogeneity compared to conventional single-modality approaches. A key example is co-detection of chromatin accessibility and gene expression that offers the opportunity to investigate the cell type-resolved gene regulatory mechanisms. Here, we described a sensitive and robust protocol of in situ SHERRY after ATAC-seq (ISSAAC-seq) for the concurrent measurement of chromatin accessibility and gene expression from the same single nucleus. The method begins with dual Tn5 tagging of open chromatin regions and DNA/RNA hybrid after reverse transcription that take place in bulk nuclei. Then various single-nucleus isolation strategies can be used based on the experimental purpose of the user. The protocol is highly modular with flexible throughputs ranging from several hundreds to tens of thousands of nuclei. The generated data are of high quality in both modalities. The entire workflow can be finished within 1 or 2 days, and the procedures works on multiple different platforms.