Preprint: Please note that this article has not completed peer review.

DART-seq

Iwijn De Vlaminck, Mridusmita Saikia, Philip Burnham, Sara H. Keshavjee, Michael F. Z. Wang, Michael Heyang, Pablo Moral-Lopez, Meleana M. Hinchman, Charles G. Danko, John S. L. Parker
DOI: 10.21203/rs.2.1655/v1

Abstract

Here we describe the step-by-step protocol of Droplet Assisted RNA Targeting by single cell sequencing (DART-seq). DART-seq allows simultaneous multiplexed amplicon sequencing and transcriptome analysis in single cells. Using a simple and quick modification of commercially available barcoded beads we have expanded the application of droplet microfluidics based high throughput single cell RNA sequencing technology to include non-polyadenylated RNA transcripts. Specific probes targeting RNA of interest were attached to the beads using a T4 DNA Ligase based enzymatic reaction.The modified DART-seq beads were then used in standard Drop-seq platform to generate single cell libraries for sequencing. We have applied DART-seq to analyze reovirus RNA sequences in infected murine L cells. As a second application we used DART-seq to investigate the B cell repertoire in human peripheral blood mononuclear cell (PBMC) samples.

Keywords
Single cell RNA sequencing, B cell repertoire, Virology

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Preprint: Please note that this article has not completed peer review.

DART-seq

Iwijn De Vlaminck, Mridusmita Saikia, Philip Burnham, Sara H. Keshavjee, Michael F. Z. Wang, Michael Heyang, Pablo Moral-Lopez, Meleana M. Hinchman, Charles G. Danko, John S. L. Parker

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Abstract

Here we describe the step-by-step protocol of Droplet Assisted RNA Targeting by single cell sequencing (DART-seq). DART-seq allows simultaneous multiplexed amplicon sequencing and transcriptome analysis in single cells. Using a simple and quick modification of commercially available barcoded beads we have expanded the application of droplet microfluidics based high throughput single cell RNA sequencing technology to include non-polyadenylated RNA transcripts. Specific probes targeting RNA of interest were attached to the beads using a T4 DNA Ligase based enzymatic reaction.The modified DART-seq beads were then used in standard Drop-seq platform to generate single cell libraries for sequencing. We have applied DART-seq to analyze reovirus RNA sequences in infected murine L cells. As a second application we used DART-seq to investigate the B cell repertoire in human peripheral blood mononuclear cell (PBMC) samples.

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

Learn more about our company.