Efficient Production of On-Target Reads for Small RNA Sequencing of Single Cells Using Modified Adapters

Ruba Khnouf, Sabrina Shore, Crystal M. Han, Jordana M. Henderson, Sarah A. Munro, Anton P. McCaffrey, Hirofumi Shintaku, Juan G. Santiago

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Although single-cell mRNA sequencing has been a powerful tool to explore cellular heterogeneity, the sequencing of small RNA at the single-cell level (sc-sRNA-seq) remains a challenge, as these have no consensus sequence, are relatively low abundant, and are difficult to amplify in a bias-free fashion. We present two methods of single-cell-lysis that enable sc-sRNA-seq. The first method is a chemical-based technique with overnight freezing while the second method leverages on-chip electrical lysis of plasma membrane and physical extraction and separation of cytoplasmic RNA via isotachophoresis. We coupled these two methods with off-chip small RNA library preparation using CleanTag modified adapters to prevent the formation of adapter dimers. We then demonstrated sc-sRNA-seq with single K562 human leukemic cells. Our approaches offer a relatively short hands-on time of 6 h and efficient generation of on-target reads. The sc-sRNA-seq with our approaches showed detection of miRNA with various abundances ranging from 16 »000 copies/cell to about 10 copies/cell. We anticipate this approach will create a new opportunity to explore cellular heterogeneity through small RNA expression.

Original languageEnglish (US)
Pages (from-to)12609-12615
Number of pages7
JournalAnalytical Chemistry
Issue number21
StatePublished - Nov 6 2018

Bibliographical note

Funding Information:
We gratefully thank Kei Iida of Medical Research Support Center of Kyoto University for a script counting reads. R.K. acknowledges support from the Jordanian-American Commission for Educational Exchange (the Binational Fulbright Commission in Jordan) through the Jordanian Visiting PostDoctoral Scholar Fellowship. C.M.H. acknowledges support from the National Institute of Standards and Technology (NIST) NRC Postdoctoral Associateship Program. C.M.H., S.A.M., and J.G.S. further acknowledge support from the NIST Joint Initiative for Metrology in Biology at Stanford. H.S. acknowledges grants from ImPACT Program of Council for Science, Technology, and Innovation (Cabinet Office, Government of Japan) and also by Japan Society for the Promotion of Science under Grants 26289035 and 26630052. Research for CleanTag was funded with a Small Business Innovation Research (SBIR) grant through the National Institutes of Health (NIH), Grant Number: 1R43HG006820-01A1. Certain commercial equipment, instruments or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology (NIST) nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Publisher Copyright:
© 2018 American Chemical Society.


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