RNA-seq library construction for small quantity of cells with engineered transposase

Abstract

Objective To build a construction strategy for RNA-seq library for small quantity of cells by optimizing Tn5 transposase assembly and purification workflow. Methods Recombinant plasmid pSUMO-Tn5 was constructed by homologous recombination. After Tn5 protein was induced to express, the product was purified with affinity chromatography followed by optimization of Tn5 assembly. Then we constructed RNA-seq library using RAW264.7 cells and analyzed the number of genes detected and the ratio of unmapped reads. Results Soluble Tn5 protein was expressed in Codon Plus BL21 with the recombinant plasmid pSUMO-Tn5. Tn5 with high purity was obtained through affinity purification. RNA-seq library for small quantity of RAW264.7 cells was successfully constructed. After the optimization of Tn5 assembly, nonspecific amplification was remarkably reduced compared with the control (P < 0.05). Conclusion Tn5 protein is effectively expressed and purified. Ultra-filtered Tn5 transposase can reduce the nonspecific amplification in RNA-seq, providing a new strategy of RNA-seq for small quantity of cells.

Keywords

• tn5 transposase
• rna-seq
• library construction