TY - JOUR
T1 - Efficient Production of On-Target Reads for Small RNA Sequencing of Single Cells Using Modified Adapters
AU - Khnouf, Ruba
AU - Shore, Sabrina
AU - Han, Crystal M.
AU - Henderson, Jordana M.
AU - Munro, Sarah A.
AU - McCaffrey, Anton P.
AU - Shintaku, Hirofumi
AU - Santiago, Juan G.
N1 - Publisher Copyright:
© 2018 American Chemical Society.
PY - 2018/11/6
Y1 - 2018/11/6
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.8b02773
DO - 10.1021/acs.analchem.8b02773
M3 - Article
C2 - 30260208
AN - SCOPUS:85054335771
SN - 0003-2700
VL - 90
SP - 12609
EP - 12615
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 21
ER -