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

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single-cell mRNA sequencing is established for cellular heterogeneity studies.
• Single-cell small RNA sequencing (sc-sRNA-seq) is challenging due to low abundance and amplification bias of small RNAs.

Purpose of the Study:

• To develop and validate novel single-cell lysis methods for enabling sc-sRNA-seq.
• To demonstrate the capability of these methods for detecting microRNA expression at the single-cell level.

Main Methods:

• Two single-cell lysis techniques were developed: chemical lysis with freezing and on-chip electrical lysis with isotachophoresis.
• These methods were coupled with off-chip small RNA library preparation using CleanTag adapters to minimize adapter dimers.
• sc-sRNA-seq was performed on single K562 human leukemic cells.

Main Results:

• The developed methods offer a short hands-on time of 6 hours and efficient generation of on-target reads.
• Successful detection of microRNA (miRNA) with a wide range of abundances (10 to 16,000 copies/cell) was achieved.
• The methods demonstrated robust performance in single K562 cell analysis.

Conclusions:

• The presented single-cell lysis approaches facilitate robust and sensitive sc-sRNA-seq.
• These methods open new avenues for exploring cellular heterogeneity via small RNA expression profiling.
• This advancement is crucial for understanding regulatory mechanisms involving small RNAs in diverse cell populations.