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Single cell lncRNAs in hematopoiesis
caution. Nevertheless, our results were in agreement with reported microarray data from 183 MDS patients, which related abnormal lncRNAs with gene expression, cancer, and malignancy.52 Also, differentially expressed lncRNAs in monosomy 7 cells were involved in similar pathways as their mRNA counterparts in our previous study.31
Our results are not a complete profile of lncRNAs due to several limitations, especially the use of only polyA- enriched RNAs,8 and the limited cell numbers from a few individuals due to the high cost of scRNA-seq. Additionally, annotation of novel lncRNAs is context dependent. We adopted commonly used pipelines,12-14,16-18 but annotation might vary using different algorithms. Nevertheless, our work creates a model for future profil- ing of the repertoire of lncRNAs in other cell types. Lineage signatures of lncRNAs are comparison-based, and thus may vary when such comparisons are made among different subsets. Others have categorized HSCs versus cells of specific lineages and among differentiated cells or distinct subsets.12-18 In contrast, we defined lncRNA signa- tures by making comparisons among subsets within a rel- atively homogeneous HSPC population, which may com- promise our power to detect differences. Furthermore, pseudotime ordering reconstructs the hematopoietic hier- archy based on bioinformatic analysis of transcriptome similarity, and it has demonstrated high agreement with purified cell compartments;44 however, dynamic gene expression in hematopoiesis might be preferably assessed in purified cell populations obtained after physical sorting based on membrane proteins, including after induction of
differentiation or other in vitro perturbations. Given the high cell-type specificity of lncRNAs, signature lncRNAs may be superior to mRNAs in discriminating and differen- tiating cell subsets or new cell types that cannot be easily distinguished based on cell surface markers. We did not compare the efficacy of lncRNAs and mRNAs in defining cell types due to a lack of detailed surface marker informa- tion for single cells. Future studies with larger cell num- bers, complete surface marker characterization, and whole transcriptome expression data should be of great interest in defining new cells/subtypes.
Rapid evolution and low species conservation are fea- tures of lncRNAs,10,11 making a human catalog a prerequi- site to successful, clinically relevant lncRNA studies. Based on next-generation sequencing and single cell tech- nology, we provide a global database that should be foun- dational for future studies of lncRNA biology in human HSPCs.
Acknowledgments
The authors acknowledge the support of the Trans-NIH Center for Human Immunology, Autoimmunity, and Inflammation (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA). We thank patients and healthy volunteers who donated bone marrow. Sequencing and technical support were provided by the DNA Sequencing and Genomics Core of NHLBI. FACS sorting was performed by Keyvan Keyvanfar and the Flow Cytometry Core of NHLBI. This research was supported by an Intramural Research Program of the National Heart, Lung, and Blood Institute.
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