Application of a Modified Smart-seq2 Sample Preparation Protocol for Rare Cell Full-length Single-cell mRNA Sequencing to Mouse Oocytes

Rebecca S Treger; Scott D Pope; Xiaojun Xing; Akiko Iwasaki

doi:10.21769/BioProtoc.3345

Application of a Modified Smart-seq2 Sample Preparation Protocol for Rare Cell Full-length Single-cell mRNA Sequencing to Mouse Oocytes

Bio Protoc. 2019 Aug 20;9(16):e3345. doi: 10.21769/BioProtoc.3345.

Authors

Rebecca S Treger¹, Scott D Pope¹, Xiaojun Xing², Akiko Iwasaki^{1

3}

Affiliations

¹ Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.
² Yale Genome Editing Center, Yale University School of Medicine, New Haven, CT 06520, USA.
³ Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.

Abstract

Endogenous retroviruses (ERV) are transposable retroelements that form ~10% of the murine genome and whose family members are differentially expressed throughout embryogenesis. However, precise regulation of ERV in germ cells remains unclear. To investigate ERV expression in oocytes, we adapted a single-cell mRNA-sequencing library preparation method to generate bulk sequencing libraries from growing oocytes in a time- and cost-efficient manner. Here, we present a modified Smart-seq2 protocol that yields full-length cDNA libraries from purified RNA obtained from low numbers of pooled immature or mature oocytes. Using this method, RNA-sequencing libraries can be generated from any rare or difficult-to-isolate populations for subsequent sequencing and retroelement expression analysis.

Keywords: Endogenous retrovirus; Oocyte; Smart-seq2; Transposable element; mRNA-sequencing.