Project description:Single cell transcriptomics has emerged as a powerful approach to dissecting phenotypic heterogeneity in complex, unsynchronized cellular populations. However, many important biological questions demand quantitative analysis of large numbers of individual cells. Hence, new tools are urgently needed for efficient, inexpensive, and parallel manipulation of RNA from individual cells. We report a simple microfluidic platform for trapping single cell lysates in sealed, picoliter microwells capable of “printing” RNA on glass or capturing RNA on polymer beads. To demonstrate the utility of our system for single cell transcriptomics, we developed a highly scalable technology for genome-wide, single cell RNA-Seq. The current implementation of our device is pipette-operated, profiles hundreds of individual cells in parallel with library preparation costs of ~$0.10-$0.20/cell, and includes five lanes for simultaneous experiments. We anticipate that this system will ultimately serve as a general platform for large-scale single cell transcriptomics, compatible with both imaging and sequencing readouts.!Series_type = Expression profiling by high throughput sequencing A microfluidic device that pairs sequence-barcoded mRNA capture beads with individual cells was used to barcode cDNA from individual cells which was then pre-amplified by in vitro transcription in a pool and converted into an Illumina RNA-Seq library. Libraries were generated from ~600 individual cells in parallel and extensive analysis was done on 396 cells from the U87 and MCF10a cell lines and from ~500 individual cells with extensive analysis on 247 cells from the U87 and WI-38 cell lines. Sequencing was done on the 3'-end of the transcript molecules. The first read contains cell-identifying barcodes that were present on the capture bead and the second read contains a unique molecular identifier (UMI) barcode, a lane-identifying barcode, and then the sequence of the transcript.
Project description:Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead-cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.
Project description:Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead-cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.
Project description:Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead-cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.
Project description:Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead-cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.
Project description:Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead-cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.
Project description:Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead-cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.
Project description:Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology. Barcode bead—cell tandems stabilized by a chemical linker are dispersed in the hydrogel in the liquid state. Upon gelation the tandems are immobilized, cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcode beads. After reverse transcription and preparation for cDNA sequencing, bioinformatic analysis reveals performance quality comparable to microfluidic-based technologies.
Project description:A widespread assumption for single-cell analyses specifies that one cell’s nucleic acids are predominantly captured by one oligonucleotide barcode. However, we show that ~13-21% of cell barcodes from the 10x Chromium scATAC-seq assay may have been derived from a droplet with more than one oligonucleotide sequence, which we call “barcode multiplets”. We demonstrate that barcode multiplets can be derived from at least two different sources. First, we confirm that approximately 4% of droplets from the 10x platform may contain multiple beads. Additionally, we find that approximately 5% of beads may contain detectable levels of multiple oligonucleotide barcodes. We show that this artifact can confound single-cell analyses, including the interpretation of clonal diversity and proliferation of intra-tumor lymphocytes. Overall, our work provides a conceptual and computational framework to identify and assess the impacts of barcode multiplets in single-cell data.
Project description:We have demonstrated in vitro transcription (IVT) of cDNA sequences from purified Jurkat T-cell mRNA immobilization on microfluidic packed beads down to single-cell quantities. The microfluidic amplified aRNA was nearly identical in length and quantity compared with benchtop reactions using the same starting sample quantities. Microarrays were used to characterize the number and population of genes in each sample, allowing comparison of the microfluidic and benchtop processes. Keywords: gene expression transcripts detected
