Project description:We developed the first droplet-based single-cell total-RNA-seq method. We refer to this platform as Multiple Annealing and Tailing-based Quantitative scRNA-seq in Droplet (MATQ-Drop). With the detection of nascent RNA species, we showed that the cell atlas of human brain samples could be effectively constructed based on nascent RNA species. Furthermore, we observed that only lncRNA species are sufficient to construct the cell atlas, suggesting that MATQ-Drop allows a large-scale identification of the cell-type-specific lncRNA species. Beyond total-RNA profiling for single nuclei, we also showed that MATQ-Drop could be used to profile the transcripts in different neuronal sub-compartments. Based on transcriptome profiling, we were able to determine different types of sub-compartments—in particular, synapses. Here we have referred to the transcriptome of individual synapses as the synaptome, and we identified different subtypes of pre-synapses and post-synapses. Furthermore, between pre-synapses and post-synapses, as well as between pre-synapses and the nuclei, we were able to identify different functional enrichments. Our result unveils unprecedented new insights about gene expression in individual synapses. It also demonstrates the feasibility of using MATQ-drop to profile the transcriptome of sub-cellular compartments.

2022-11-30 | GSE174293 | GEO

High-Throughput Transcriptome Profiling of Single Nuclei and Single Synapses Using Single-Cell Total-RNA-Seq [Mm]

2022-11-30 | GSE199344 | GEO

Scalable co-sequencing of RNA and DNA from individual nuclei

Project description:We present DEFND-seq (DNA and Expression From Nucleosome Depletion), a scalable method for co-sequencing RNA and DNA from single nuclei. In DEFND-seq we treat nuclei with lithium diiodosalicylate to disrupt chromatin and expose genoimc DNA. The nuclei are then tagmented with Tn5 transposase, which fragments and tags gDNA. Tagmented nuclei are loaded into a microfluidic droplet generator which combines nuclei, beads containing transcriptomic and genomic barcodes, and reverse transcription reagents into single droplets. Ultimately two libraries are created, one for nuclear mRNA and one for genomic DNA, with each library containing barcodes linking it to its nuclei of origin, thus allowing simultaneous analysis of single nuclei transcriptomes and genomes. Once nuclei have been depleted of nucleosomes, all steps can be performed using a 10x Chromium Controller and 10x Multiome Kit without further experimental modification.

2023-02-09 | GSE224149 | GEO

Dissecting cell-type composition and activity-dependent transcriptional state in mammalian brains by massively parallel single-nucleus RNA-Seq

Project description:Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues, such as adult mammalian brains, is challenging. Here, we integrate sucrose-gradient assisted nuclei purification with droplet microfluidics to develop a highly scalable single-nucleus RNA-Seq approach (sNucDrop-Seq), which is free of enzymatic dissociation and nuclei sorting. By profiling ~18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-Seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity, but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, in vivo.

2017-12-07 | GSE106678 | GEO

Cell-type-specific Brain Methylomes Profiled via Ultralow-input Microfluidics

Project description:Methylomic studies require substantial amounts of DNA samples and this restriction hinders applications involving scarce animal or patient samples with direct biomedical relevance. Here we report a microfluidics-based reduced representative bisulfite sequencing protocol, MIcrofluidic Diffusion-based RRBS (MID-RRBS), that permits methylomic profiling with sub-1 ng starting DNA. Using this technology, we studied DNA methylation in NeuN+ and NeuN- fractions isolated from mouse cerebellum, revealing cell-type specific methylomic patterns. We also studied the DNA methylation in NeuN+ nuclei isolated from clozapine or vehicle treated mouse frontal cortex.

2018-02-15 | GSE88716 | GEO

MicDrop: Droplet Microfluidic Assay for Bacteria

Project description:High-throughput isolation and culture of human gut bacteria with droplet microfluidics

| PRJEB33065 | ENA