A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples

Abstract Background RNA-sequencing (RNA-seq) has emerged as one of the most sensitive tool for gene expression analysis. Among the library preparation methods available, the standard poly(A) + enrichment provides a comprehensive, detailed, and accurate view of polyadenylated RNAs. However, on samples of suboptimal quality ribosomal RNA depletion and exon capture methods have recently been reported as better alternatives. Methods We compared for the first time three commercial Illumina library preparation kits (TruSeq Stranded mRNA, TruSeq Ribo-Zero rRNA Removal, and TruSeq RNA Access) as representatives of these three different approaches using well-established human reference RNA samples from the MAQC/SEQC consortium on a wide range of input amounts (from 100 ng down to 1 ng) and degradation levels (intact, degraded, and highly degraded). Results We assessed the accuracy of the generated expression values by comparison to gold standard TaqMan qPCR measurements and gained unprecedented insight into the limits of applicability in terms of input quantity and sample quality of each protocol. We found that each protocol generates highly reproducible results (R 2 > 0.92) on intact RNA samples down to input amounts of 10 ng. For degraded RNA samples, Ribo-Zero showed clear performance advantages over the other two protocols as it generated more accurate and better reproducible gene expression results even at very low input amounts such as 1 ng and 2 ng. For highly degraded RNA samples, RNA Access performed best generating reliable data down to 5 ng input. Conclusions We found that the ribosomal RNA depletion protocol from Illumina works very well at amounts far below recommendation and over a good range of intact and degraded material. We also infer that the exome-capture protocol (RNA Access, Illumina) performs better than other methods on highly degraded and low amount samples

A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte

The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury–repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the ‘pulmonary ionocyte’, which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium

Additional file 1: Figure S1. of A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples

Bioanalyzer profile of the fragment size distribution for the intact SEQC-A and SEQC-B samples. The curve for SEQC-A is shown in red and the curve for SEQC-B in blue. The two peaks represent the intact 18S and 28S ribosomal RNA profiles. Figure S2. Bioanalyzer profile of the fragment size distribution for the degraded SEQC-A and SEQC-B samples. The curve for SEQC-A is shown in red and the curve for SEQC-B in blue. The peaks for the 18S and 28S ribosomal RNAs are now following a unimodal distribution with a much wider peak around a fragment size of 850 nt, reflecting the level of degradation. Figure S3. Bioanalyzer profile of the fragment size distribution for the highly-degraded SEQC-A and SEQC-B samples. The curve for SEQC-A is shown in red and the curve for SEQC-B in blue. The peaks for the 18S and 28S ribosomal RNAs are now following a unimodal distribution with a much wider peak around a fragment size of 150–200 nt, reflecting a high level of degradation. Figure S4. Bargraph of the alignment statistics for the SEQC-B sample and all three protocols. Each bar represents the averaged values across the three technical replicates per condition. The percentage of total aligned reads is represented by the height of the bar, and the percentage of reads aligning to exons is in red, introns in blue, and intergenic regions in green. Figure S5. Venn diagram of the protein coding genes detected by each of the three protocols. Venn diagram of the protein coding genes detected by each of the three protocols on degraded samples at the input amounts 10 ng for RNA Access and 100 ng for Ribo-Zero and TruSeq. A gene is considered “expressed” if it has a FPKM value of at least 0.3 in one of the three technical replicates of at least one of the two samples (SEQC-A or SEQC-B). Table S1. Simplified Ensembl gene type mapping. The original Ensembl (v76) gene type category is contained in the left column and the simplified category is contained in the right column. (PDF 661 kb

Mir-210 promotes sensory hair cell formation in the organ of Corti

Background: Hearing loss is the most common sensory defect with several hundred million people worldwide having hearing disorders. In most cases, the cause of hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. However, despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration and thereby provide new avenues for the treatment of hearing loss. Results: In order to identify new genetic elements enabling regeneration of sensory hair cells in the inner ear, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent microRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on miRSeq we selected eight most differentially expressed miRNAs for further characterization. Of those, miR-210 knock-down in vitro resulted in hair cell marker expression in UB-OC1, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. By using a lineage tracing mouse model, we identified transdifferentiation of supporting epithelial cells as the likely mechanism for new hair cell formation. Potential miR-210 targets were predicted in silico and identified experimentally using a miR-trap approach. Conclusion: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells. Our data suggest that miR-210 might be a potential new factor for hearing loss therapy. Moreover, identification of inner ear pathways regulated by miR-210 identified novel drug targets for the treatment of hearing loss

A Simple and Efficient CRISPR Technique for Protein Tagging

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells

Single-cell and bulk transcriptomics of the liver reveals potential targets of NASH with fibrosis.

Fibrosis is characterized by the excessive production of collagen and other extracellular matrix (ECM) components and represents a leading cause of morbidity and mortality worldwide. Previous studies of nonalcoholic steatohepatitis (NASH) with fibrosis were largely restricted to bulk transcriptome profiles. Thus, our understanding of this disease is limited by an incomplete characterization of liver cell types in general and hepatic stellate cells (HSCs) in particular, given that activated HSCs are the major hepatic fibrogenic cell population. To help fill this gap, we profiled 17,810 non-parenchymal cells derived from six healthy human livers. In conjunction with public single-cell data of fibrotic/cirrhotic human livers, these profiles enable the identification of potential intercellular signaling axes (e.g., ITGAV-LAMC1, TNFRSF11B-VWF and NOTCH2-DLL4) and master regulators (e.g., RUNX1 and CREB3L1) responsible for the activation of HSCs during fibrogenesis. Bulk RNA-seq data of NASH patient livers and rodent models for liver fibrosis of diverse etiologies allowed us to evaluate the translatability of candidate therapeutic targets for NASH-related fibrosis. We identified 61 liver fibrosis-associated genes (e.g., AEBP1, PRRX1 and LARP6) that may serve as a repertoire of translatable drug target candidates. Consistent with the above regulon results, gene regulatory network analysis allowed the identification of CREB3L1 as a master regulator of many of the 61 genes. Together, this study highlights potential cell-cell interactions and master regulators that underlie HSC activation and reveals genes that may represent prospective hallmark signatures for liver fibrosis

Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening.

Correlation of sgRNA library distribution between technical replicates of HCC-827 and CHP-212 cells. (XLSX 30 kb

Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology

Oral and intestinal mucositis is a debilitating, often dose limiting side effect of radiation treatment. A mouse model of mucositis, induced by gamma irradiation, leads to weight loss and tissue damage, similar to that observed in patients. This model reflects the human ailment as it responds to keratinocyte growth factor (KGF), the standard of care treatment. Culturing of intestinal crypt organoids derived from primary cells allowed the development of a 3D assay to monitor the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay closely resembles the mouse model as KGF and Roof Plate-Specific Spondin-1 (RSPO1) enhanced the recovery of crypt organoids following radiation. Screening identified tool compounds that increased the survival of organoids post radiation. Repeated testing of these compounds revealed that the organoids changed their response over time. To investigate this adaptive behavior, intestinal organoid cultures were studied over time. Samples of organoids at various time points were used to prepare mRNA for unbiased transcriptome analyses. This expression profiling revealed a number of genes and pathways that were modulated over time, providing a rationale for the altered sensitivity of the intestinal crypt organoid cultures. This report describes the development of an in vitro assay that reflects the response of disease to therapeutic treatment. The assay was miniaturized and used to identify bioactive tool compounds, which served as probes to interrogate the patho-physiology of organoids over prolonged culture conditions. In vitro disease models based on primary 3D cell cultures represent valuable tools to identify potential drug targets and bioactive hits

Large-scale functional epigenomic screens reveal cancer lineage-specific regulation of YAP responsive elements

YAP and TAZ are potent transcriptional co-factors engaging TEAD proteins downstream of Hippo signaling. Malignant pleural mesothelioma (MPM) and uveal melanoma (UM) are distinct cancer lineages bearing different genetic aberrations that ultimately lead to YAP activation. Here we use MPM and UM as prototypical cancers displaying, respectively, Hippo-dependent and -independent YAP activation to demonstrate that, while YAP is essential in both diseases, its interaction with TEAD is dispensable in UM, potentially limiting the application of TEAD inhibitors. Large scale functional epigenomic screens of YAP regulatory elements in MPM and UM reveal: 1) lineage-specific enhancers controlling broad oncogene dependencies (e.g., MYC) in both diseases, 2) rewiring of MAPK transcriptional regulatory networks in MPM, translating into synergistic efficacy of TEAD and MAPK inhibitors and 3) enrichment of melanocytic master regulators at functional YREs in UM. Our work prompts the design of tailored therapeutic strategies to inhibit YAP signaling in specific cancers