HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes

Marshall, Jamie L.; Doughty, Benjamin R.; Subramanian, Vidya; Guckelberger, Philine; Wang, Qingbo; Chen, Linlin M.; Rodriques, Samuel G.; Zhang, Kaite; Fulco, Charles P.; Nasser, Joseph; Grinkevich, Elizabeth; Noel, Teia; Mangiameli, Sarah; Bergman, Drew T.; Greka, Anna; Lander, Eric S.; Chen, Fei; Engreitz, Jesse M.

doi:10.1073/pnas.2010738117

Cited by 28 publications

(16 citation statements)

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“…However, the use of poly (A) tagging strategy improved the efficiency of conversion to a mRNA molecule to amplified cDNA, and used unique molecular identifiers (UMI) tags for each cell can increase the detection of low expression genes [ 19 ]. Although the sequencing technology based on probe hybridization can improve the detection number of cells and transcript detection sensitivity, probe hybridization requires multiple cleaning, resulting in the loss of some cells [ 20 ]. Therefore, different single-cell technologies have different limitations.…”

Section: Discussionmentioning

confidence: 99%

“…However, cell lysis, mRNA capture, and reversed transcription can be efficiently carried out by improving the parameters of the microfluidic system, such as fluid speed and pressure [ 17 – 19 ]. In addition, hybridization of probes to RNA for sequencing (HyPR-seq), which can easily detect more than 100,000 cells in a single experiment, and achieve high sensitivity for individual transcripts in single cells and low-abundance transcripts [ 20 ]. However, multiple rounds of washes for probe hybridization and ligation, which result in some cell loss.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

et al. 2021

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Background Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing. Results Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified. Conclusions The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

et al. 2021

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“…Organ-specific and cell-type specific transcriptomic aging clocks are also a possibility, to invoke some integrated measure of cellular age as a readout ( Meyer and Schumacher 2021 ). The sensitivity of single-cell transcriptomics to small variations in gene expression, or to low abundance transcripts, is considerably lower than that of bulk transcriptomics, but targeted sequencing approaches like HyPR-Seq can achieve much greater sensitivity for a defined, yet still diverse subset of genes, such as aging clock gene arrays ( Marshall et al, 2020 ). More complex clock-like signatures computed from comparisons across species or from responses to known aging drugs ( Tyshkovskiy et al, 2019 ) could also be used as readouts for pooled screening.…”

Section: Applications To Aging Biologymentioning

confidence: 99%

In vivo Pooled Screening: A Scalable Tool to Study the Complexity of Aging and Age-Related Disease

Jensen

Marblestone

2021

Front. Aging

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Biological aging, and the diseases of aging, occur in a complex in vivo environment, driven by multiple interacting processes. A convergence of recently developed technologies has enabled in vivo pooled screening: direct administration of a library of different perturbations to a living animal, with a subsequent readout that distinguishes the identity of each perturbation and its effect on individual cells within the animal. Such screens hold promise for efficiently applying functional genomics to aging processes in the full richness of the in vivo setting. In this review, we describe the technologies behind in vivo pooled screening, including a range of options for delivery, perturbation and readout methods, and outline their potential application to aging and age-related disease. We then suggest how in vivo pooled screening, together with emerging innovations in each of its technological underpinnings, could be extended to shed light on key open questions in aging biology, including the mechanisms and limits of epigenetic reprogramming and identifying cellular mediators of systemic signals in aging.

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“…The ability to sort and recover cells based on their RNA content could allow selective recovery of cell types of interest that cannot be isolated via FACS, including rare cell types revealed via single-cell transcriptomic studies 35 , cells containing latent integrated viruses, 36 and cells expressing RNA splice isoforms or noncoding RNAs. 37 While RT-PCR for single cells within encapsulated FACS-sortable double emulsion (DE) droplets provides a promising potential sorting method, [11][12][13][14][15][16][17][18] realizing this technology requires successful RT-PCR from single cells within small volumes (<100 pL), at odds with many prior publications reporting that singlecell RT-PCR cannot take place at volumes <1 nL.…”

Section: Discussionmentioning

confidence: 99%

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

Khariton

McClune

Brower

et al. 2022

Preprint

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Microfluidic droplet assays enable single-cell PCR and sequencing assays at unprecedented scale, with most methods encapsulating cells within nanoliter sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) droplets allows sorting on commercially available FACS machines, making it possible to isolate single cells based on phenotypes of interest for downstream sequencing and PCR analyses. However, DE droplets must be within the range of large cells (<20 pl) for sorting on standard cytometers, posing challenges for molecular biology as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, but that this cleavage can be efficiently alleviated through heat lysis that simultaneously inactivates inhibitory lysate components. We further show that the magnitude of RT-PCR inhibition depends strongly on cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL double emulsion droplets with fluorescence detectable via FACS. These results open up exciting new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet based single-cell analysis technologies.

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HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes

Cited by 28 publications

References 50 publications

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

In vivo Pooled Screening: A Scalable Tool to Study the Complexity of Aging and Age-Related Disease

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

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