Expanding the Chinese hamster ovary cell long noncoding RNA transcriptome using RNASeq

Abstract

Our ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade following the development of next generation sequencing technology and publication of reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small noncoding RNAs. The annotation of long noncoding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high‐resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHO K1 genome. In addition, the utilisation of strand‐specific sequencing enabled the identification of more than 1,000 new antisense and divergent lncRNAs. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community.

A simple guide to de novo transcriptome assembly and annotation

“…mRNA or rRNA) and have a low coding potential must be lncRNAs. We direct the interested reader to consult Motheramgari et al [ 144 ] and Kashyap et al [ 145 ] for demonstrations of elimination techniques for classifying lcnRNAs.…”

Tracing production instability in a clonally derived CHO cell line using single‐cell transcriptomics

“…RNA‐seq has also been utilized to characterize changes not only in gene expression (Clarke et al, 2019; Sha et al, 2018) but also in alternative splicing of CHO cell mRNAs (Tzani et al, 2020). The technology has also played a crucial role in the annotation of noncoding RNA molecules such as microRNAs (Hackl et al, 2012) and long noncoding RNAs (Hernandez et al, 2019; Motheramgari et al, 2020; Vito et al, 2020). As our knowledge of the CHO cell biological system becomes increasingly sophisticated, precise routes for genetic engineering are being identified and accurate genome‐scale models for the prediction of cell line characteristics are being developed (Gutierrez et al, 2020).…”

Subcloning induces changes in the DNA‐methylation pattern of outgrowing Chinese hamster ovary cell colonies

“…[4] As a specific phenotype is not likely to be determined by a single mutation or the change in expression of a single gene, the combination of genetic and epigenetic variation actually provides cells with a wide space of possible transcriptomes and resulting phenotypes across the 10-15,000 expressed coding and the even more non-coding genes. [19,38,56,57] This wide space of variation in transcript expression levels would explain the known adaptability of CHO cells and the success of process and cell line development that the biopharmaceutical industry has seen over the last 30 years.…”