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

Abstract

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.

Figure 1

Single-cell lyses for sc-sRNA-seq with CleanTag library preparation: (A) lysis via 0.13% Triton X-100 and freezing overnight (∼12 h) and (B) electrical lysis and ITP-based extraction of cytoplasmic RNA (∼5 min). Both samples are processed with CleanTag small RNA library prep (6 h).

Figure 2

Capillary gel electropherograms using the Agilent Bioanalyzer for products of the CleanTag library preparation. (A) Electropherogram for single K562 cell lysed via Triton X-100 with overnight-freezing and for (B) on-chip ITP extracted single K562 cell cytoplasmic RNA. Both electropherograms show abundant content ranging from 120 to 150 nt, consistent with ligated mature small RNA molecules.

Figure 3

Composition of sequencing reads obtained with two lyses protocols, (A) Triton and (B) microfluidics-based approaches (single K562 cells), and (C) that obtained with data from Faridani et al. (single HEK293FT cells).

Figure 4

Characterization of sc-sRNA-seq approaches using single K562 cells: (A) number of precursor RNAs (pre-miRNAs, tRNAs, snoRNAs) and (B) number of mature sRNAs (miRNAs, tsRNAs, and sdRNAs) detected with Triton and microfluidic-based lyses and single-cell-quantity total RNA (10 pg) extracted from K562 cells. (C–E) Length distribution of aligned reads on miRNA, tRNA, and snoRNA. (F) Pearson correlation in all combinations of replicate sample pairs prepared with the same method. (G) Pearson correlation of sRNA expression to control experiments with 100 ng of total RNA.

Figure 5

Scatter plots of sRNA expression comparing two lysing and extraction methods to 10 pg of K562 prepurified RNA sample. (A) Plotted are correlation of sRNA expression for Triton vs 10 pg total RNA for small RNA types (miRNA, tsRNA, sdRNA, lincRNA, and snRNA). (B) Correlation of sRNA expression of microfluidics-based approach vs 10 pg total RNA for the same small RNA types. The Triton samples were better correlated with 10 pg total RNA especially when comparing nuclear enriched sRNA such as sdRNA and snRNA since the microfluidics-based approach used only cytoplasmic fraction. Note that DEG is a database of essential genes.

Figure 6

Expression pattern of miRNAs known to be expressed in K562 cells for the following samples: (A) single K562 cells prepared with Triton X-100-based lysis, (B) those prepared with microfluidics-base lysis and extraction, (C) 10 pg of total RNA of K562 cells, and (D) 100 ng of total RNA of K562 cells. miRNA sequences are listed in the order of increasing median abundance as observed in the 100 ng of total RNA data (see also White et al. for comparison). The 100 ng of total RNA showed the least variation as expected.