Small-seq for single-cell small-RNA sequencing

Abstract

Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from individual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs); however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2–3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.

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Fig. 1: Outline of the Small-seq library preparation protocol.
Fig. 2: Size selection by implementation of the crush and soak method.
Fig. 3: Expected results after Bioanalyzer profiling of the library pools.

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Acknowledgements

We thank B. Pannagel at the CMB FACS facility at Karolinska Institutet for performing the single-cell sorting. This research was supported by grants from the Swedish Research Council (grant no. 2017-01062 to R.S.) and the Bert L. & N. Kuggie Vallee Foundation (to R.S.).

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Authors

Contributions

M.H.-J. developed and optimized the protocol, analyzed the data, and prepared the manuscript. I.A. constructed the bioinformatics pipeline, analyzed the data, and prepared the manuscript. R.S. supervised the development of the computational analyses and prepared the manuscript. O.R.F. conceived the study, developed the method, interpreted the results, and prepared the manuscript.

Corresponding author

Correspondence to Omid R Faridani.

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Competing interests

O.R.F. and R.S. have filed a patent application (PCT/US2017/037620) on the protocol for single-cell small-RNA sequencing. The remaining authors declare no competing interests.

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Key reference using this protocol

1. Faridani, O. R. et al. Nat. Biotechnol. 34, 1264–1266 (2016): https://doi.org/10.1038/nbt.3701

Integrated supplementary information

Supplementary Figure 1 Number of genes and molecules in a test example.

Number of captured small RNA–encoding genes (a) and small RNA molecules (b) in libraries containing the rRNA-masking oligonucleotide with HEK cells or without cells.

Supplementary Figure 2 Outline of the gating strategy implemented for sorting HEK293FT single cells.

Gates for selecting single cells are shown in plots for Side Scatter Area (SSC-A, logarithmic) against Forward Scatter Area (FSC-A) (a), Forward Scatter Width (FSC-W) against Forward Scatter Area (FSC-A) (b), and Side Scatter Width (SSC-W) against Side Scatter Area (SSC-A, logarithmic) (c). Propidium iodide was applied to stain for dead cells (d).

Supplementary Figure 3 Percentage of filtered and genome-mapped reads.

Libraries obtained from HEK cells or no cells (all with the rRNA-masking oligonucleotide) were generated and sequenced. Here, the percentages are shown for sequenced reads that are filtered (grey), cannot be mapped (green), are mapped to multiple loci (blue), and are uniquely mapped (purple).

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Supplementary Figures 1–3 and Supplementary Table 1

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Hagemann-Jensen, M., Abdullayev, I., Sandberg, R. et al. Small-seq for single-cell small-RNA sequencing. Nat Protoc 13, 2407–2424 (2018). https://doi.org/10.1038/s41596-018-0049-y

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