Sensitive and Low-Bias Transcriptome Sequencing Using Agarose PCR

Transcriptome sequencing has emerged
as an important research tool
for exploring the mysteries of life at the single-cell level. However,
its wide application is limited by the bias associated with the amplification
reactions which is essential for library building of trace RNA. In
this study, low-melting-point agarose was added to the amplification
reactions to take advantage of its molecular crowding effect and polymer
cross-linked structure to improve the sensitivity of the reactions
and reduce bias. To further evaluate the performance of the method,
it was applied to transcriptome sequencing of microregion samples
from brain tissue sections of mice with Parkinson’s disease
at the single cell level. The results showed that agarose PCR had
better performance than in-tube PCR. Further application of agarose
PCR to transcriptome library sequencing could obtain data closer to
that of unamplified. With the addition of low melting point agarose,
the sensitivity of the amplification reaction was significantly increased,
while homogeneity was increased by approximately 2-fold. Not only
that, but this work also provides 11% sensitivity improvement for
spatial transcriptomic study on Parkinson’s disease-associated
gene detection. The agarose PCR provides a new tool for efficient
and homogeneous amplification of trace samples and can be widely used
for spatial transcriptome library sequencing and studies.