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Seq-Well: A portable, low-cost platform for single-cell RNA-seq of low-input samples

ABSTRACT: Seq-Well: A portable, low-cost platform for single-cell RNA-seq of low-input samples

OTHER RELATED OMICS DATASETS IN: GSE92495

PROVIDER: PRJNA374551 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5250850_1.fastq.gz	Fastqsanger.gz
	SRR5250850_2.fastq.gz	Fastqsanger.gz
	SRR5250841_1.fastq.gz	Fastqsanger.gz
	SRR5250841_2.fastq.gz	Fastqsanger.gz
	SRR5250851_1.fastq.gz	Fastqsanger.gz

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Seq-Well: A portable, low-cost platform for single-cell RNA-seq of low-input samples

Project description:Single-cell RNA -seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA -seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We characterize Seq-Well extensively and use it to profile thousands of primary human macrophages exposed to tuberculosis.

2017-02-17 | GSE92495 | GEO

Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols

Project description:Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols

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Systematic comparative analysis of strand-specific, cost-effective, RNA-seq library preparation methods for low input samples

Project description:Despite the precipitous decline in the cost of genome sequencing over the last few years, library preparation for RNA-seq is still laborious and expensive for high throughput screening for drug discovery. Limited availability of RNA generated by some experimental workflows poses an additional challenge and typically adds to the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that also maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies – Swift and Swift Rapid – using the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics, as our reference. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark differential gene expression in these kits, at input quantities ranging between 10 ng to 500 ng. Read quality and alignment metrics revealed high mapping efficiency and uniform read coverage through genes for all samples across all three kits. Normalized read counts between all treatment groups were in high agreement, with pairwise Pearson correlation coefficients >0.97. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits are cost effective and offer shorter workflow times enabled by their patented Adaptase technology. Furthermore, the Swift RNA kit allows for a relatively broader (and lower) input range, producing consistent results across diverse samples. The Swift Rapid RNA method is the fastest and most cost effective NGS workflow that is best suited for higher RNA yields, with the exact same RNA input range as the Illumina TruSeq kit. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways attributable to input mRNA concentration.

2021-02-23 | GSE167300 | GEO

A scalable, low cost, multi-omic interrogation and sample hashing workflow for single-cell analysis using the Seq-Well S3 platform

Project description:Clinical samples promise unparalleled insights into the cellular mechanisms that underlie pathological conditions and therapeutic responses. However, they often can be precious, with few cells available for single-cell analysis, necessitating effort to maximize the amount of information that can be garnered from each. Here, we introduce a low material input, cost-effective protocol for conducting multi-omic analyses and sample hashing on single-cell suspensions using the Seq-Well S3 platform. Our protocol, designed to be both accessible and affordable, leverages readily available reagents and standard laboratory equipment, significantly lowering barriers to entry for researchers in low- and middle-income countries. The method detailed herein offers a streamlined and efficient workflow for: (1) precise staining of single-cell suspensions with antibody-oligo conjugates for accurate cell surface protein identification and effective sample multiplexing; (2) reliable generation of Seq-Well S3 sequencing libraries; (3) optional generation of bulk-RNA sequencing libraries through an optimized SMART-seq2 protocol; and, (4) robust computational pipelines for in-depth multi-omic data analysis. This protocol works with fragile and limited cell inputs (here, outlined for 15,000 cells per 200 µL - a fraction of the input required by most commercial methods). It also offers significant cost and time savings, with the entire process from cell isolation to sequencing taking only 3-6 days, plus an additional 1-2 days for data processing. In sum, this generally applicable pipeline empowers researchers around the globe to apply single-cell multiomics to advance their own research agendas.

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2020-10-22 | GSE149666 | GEO

LiBis: An ultrasensitive alignment method for low-input bisulfite sequencing

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Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols

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