Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues.

ABSTRACT:

Background

Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing.

Results

Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 ge

SUBMITTER: Jia E 

PROVIDER: S-EPMC8579666 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature