Single-nuclei RNA-seq on human retinal tissue provides improved transcriptome profiling

Abstract

26Gene expression profiling is an effective way to provide insights into cell function. 27 However, for heterogeneous tissues, bulk RNA-Seq can only provide the average gene 28 expression profile for all cells from the tissue, making the interpretation of the sequencing 29 result challenging. Single-cell RNA-seq, on the other hand, generates transcriptomic 30 profiles of individual cell and cell types, making it a powerful method to decode the 31 heterogeneity in complex tissues. 32 The retina is a heterogeneous tissue composed of multiple cell types with distinct 33 functions. Here we report the first single-nuclei RNA-seq transcriptomic study on human 34 neural retinal tissue to identify transcriptome profile for individual cell types. Six retina 35 samples from three healthy donors were profiled and RNA-seq data with high quality was 36 obtained for 4730 single nuclei. All seven major cell types were observed from the dataset 37 and signature genes for each cell type were identified by differential gene express 38 analysis. The gene expression of the macular and peripheral retina was compared at the 39 cell type level, showing significant improvement from previous bulk RNA-seq studies. 40Furthermore, our dataset showed improved power in prioritizing genes associated with 41 human retinal diseases compared to both mouse single-cell RNA-seq and human bulk 42 RNA-seq results. In conclusion, we demonstrated that feasibility of obtaining single cell 43 transcriptome from human frozen tissues to provide additional insights that is missed by 44 either the human bulk RNA-seq or the animal models. 45 46 48 cell type and state, and investigating human diseases 1-3 . Transcriptome can be more 49 powerful when combined with other 'omics' data to build prediction models on human 50 diseases 4 . For example, by combining transcriptomic data and proteomic data, a list of 51 candidate disease genes can be predicted with high specificity 5 . However, until recently, 52 vast majority transcriptome profiles are generated from profiling tissue samples 53 containing thousands to millions of cells. Thus, gene expression information of individual 54 cells would be lost. For tissues with high cellular heterogeneity, knowing the transcriptome 55 profiles of each cell type would be important for both identification of novel cell types and 56 understanding the functional organization of the tissue. Cell sorting would be required to 57 obtain transcriptome of a single cell type; not only was it not always practical, but also the 58 heterogeneities of many tissues were not fully revealed. This gap was met by the 59 development of the high throughput single-cell RNA-seq technology 6-8 . 60 61 Transcriptomic studies on the single cell level was first performed decades ago 9,10 , while 62 the first single-cell transcriptome study based on Next-Generation Sequencing was 63 reported ten years ago 11 . Since then, technologies have been dramatically improved in 64 scale and sensitivity. Development in single-cell isolation, such as microf...

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids

“…Single-cell transcriptomes of human retina have not been reported. We decided to profile single-cell transcriptomes of well-characterized fresh normal human macula (46,47) using single-nucleus RNA-seq as a surrogate (referred to as single-cell transcriptomes hereafter for convenience) for these reasons: (i) Fresh human retinal tissues are not readily available, (ii) cell nuclei are well preserved in frozen human retinal tissues, and (iii) single-nucleus RNA-seq is highly correlated to scRNA-seq (48). As described in Materials and Methods, single-cell transcriptomes of human macula were successfully generated, resulting in cell clusters of cones, rods, bipolar cells, MG cells, and interneurons (SI Appendix, Fig.…”

Single cell RNA sequencing reveals cellular diversity of trisomy 21 retina

“…We noticed that two major retinal cell types (horizontal cells and amacrine cells) were undetected in our data, probably because of their relatively low proportion. The cell type proportion of the sample (Fig 2b) demonstrated the abnormal constitution of retina neuron compared with normal fetal at the same gestational weeks according to recent researches 11,17 .…”

A single-cell transcriptome atlas of the aging human and macaque retina

“…By profiling 119,520 single cells, we provided detailed single-cell transcriptomes of adult and aged primate retinae with regional information. The retinal transcriptome at single-cell or single-nuclei level has been reported [3,[9][10][11][12][13][14].…”

“…The molecular differences between the foveal and peripheral retina are important for understanding human visual function. To date, multiple analyses in primates have demonstrated the regionspecific retinal transcriptomes in developing and adult foveal and peripheral retina, suggesting distinct transcriptional regulations in the two regions [3,[9][10][11][12][13][14].…”