FLED: a full-length eccDNA detector for long-reads sequencing data.

Reconstructing the full-length sequence of extrachromosomal circular DNA (eccDNA) from short sequencing reads has proved challenging given the similarity of eccDNAs and their corresponding linear DNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length eccDNAs. Herein, a novel algorithm was developed, called Full-Length eccDNA Detection (FLED), to reconstruct the sequence of eccDNAs based on the strategy that combined rolling circle amplification and nanopore long-reads sequencing technology. Seven human epithelial and cancer cell line samples were analyzed by FLED and over 5000 full-length eccDNAs were identified per sample. The structures of identified eccDNAs were validated by both Polymerase Chain Reaction (PCR) and Sanger sequencing. Compared to other published nanopore-based eccDNA detectors, FLED exhibited higher sensitivity. In cancer cell lines, the genes overlapped with eccDNA regions were enriched in cancer-related pathways and cis-regulatory elements can be predicted in the upstream or downstream of intact genes on eccDNA molecules, and the expressions of these cancer-related genes were dysregulated in tumor cell lines, indicating the regulatory potency of eccDNAs in biological processes. The proposed method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length eccDNA sequences. FLED is implemented using Python3 which is freely available on GitHub (https://github.com/FuyuLi/FLED).

PLAU promotes cell proliferation and migration of head and neck cancer via STAT3 signaling pathway.

It was reported that within the head and neck cancer (HNC) cell line CAL21 the epithelial-mesenchymal transition (EMT) and cell proliferation were promoted by Urokinase-Type Plasminogen Activator (PLAU) proteinase through TNFRSF12A. Additionally, in this paper HNC cell lines refer to Fadu and Tu686. A novel PLAU-STAT3 axis was found to be involved in HNC cell line proliferation and metastasis. PLAU expression in HNC samples was upregulated, besides, the elevated expression of PLAU was linked to the lower overall survival (OS) and disease-free survival (DFS). Ectopic PLAU expression promoted cell proliferation and migration, while PLAU knockdown exhibited opposite results. RNA-seq data identified the JAK-STAT signaling pathway, confirmed by western blotting. A recovery assay using S3I-201, a selective inhibitor of signal transducer and activator of transcription 3 (STAT3), indicated that PLAU promoted HNC cell line progression via STAT3 signaling in vitro. The oncogenic role of PLAU in HNC tumor growth in vivo was confirmed using xenograft models. In summary, we identified the tumorigenic PLAU function in the HNC progress. PLAU may represent a potential prognostic biomarker of HNC and the PLAU-STAT3 pathway might be considered a therapeutic target of HNC.

Osr1-mediated mesothelial transition of liver mesenchymal cells exacerbates fibrotic liver damage.

In chronic liver diseases, hepatic stellate cells (HSCs) are induced to form the myofibroblasts responsible for scar formation, leading to liver fibrosis and cirrhosis. Here, single-cell RNA sequencing with in vivo lineage tracing in nonalcoholic steatohepatitis (NASH) model mice reveals a subpopulation of HSCs transitioning back to a state resembling their developmental precursors, mesothelial cells (MCs), after liver injury. These damage-associated intermediates between HSCs and MCs (DIHMs) can be traced with a dual recombinase system by labeling Krt19-expressing cells within prelabeled Pdgfrb+ HSCs, and DIHMs highly express inflammation- and fibrosis-associated genes. Cre and Dre-inducible depletion of DIHMs by administering diphtheria toxin reduces liver fibrosis and alleviates liver damage in NASH model mice. Importantly, knockdown of Osr1, a zinc finger transcription factor of the OSR gene family, can block DIHM induction in vitro. Conditional knockout Osr1 in Pdgfrb-expressing mesenchymal cells in NASH model mice can reduce liver fibrosis in vivo. Our study collectively uncovers an injury-induced developmental reversion process wherein HSCs undergo what we call a mesenchymal-to-mesothelial transition, which can be targeted to develop interventions to treat chronic liver diseases.

Human induced pluripotent stem cell/embryonic stem cell-derived pyramidal neuronal precursors show safety and efficacy in a rat spinal cord injury model.

Nerve regeneration and circuit reconstruction remain a challenge following spinal cord injury (SCI). Corticospinal pyramidal neurons possess strong axon projection ability. In this study, human induced pluripotent stem cells (iPSCs) were differentiated into pyramidal neuronal precursors (PNPs) by addition of small molecule dorsomorphin into the culture. iPSC-derived PNPs were transplanted acutely into a rat contusion SCI model on the same day of injury. Following engraftment, the SCI rats showed significantly improved motor functions compared with vehicle control group as revealed by behavioral tests. Eight weeks following engraftment, the PNPs matured into corticospinal pyramidal neurons and extended axons into distant host spinal cord tissues, mostly in a caudal direction. Host neurons rostral to the lesion site also grew axons into the graft. Possible synaptic connections as a bridging relay may have been formed between host and graft-derived neurons, as indicated by pre- and post-synaptic marker staining and the regulation of chemogenetic regulatory systems. PNP graft showed an anti-inflammatory effect at the injury site and could bias microglia/macrophages towards a M2 phenotype. In addition, PNP graft was safe and no tumor formation was detected after transplantation into immunodeficient mice and SCI rats. The potential to reconstruct a neuronal relay circuitry across the lesion site and to modulate the microenvironment in SCI makes PNPs a promising cellular candidate for treatment of SCI.

Solution Synthesis and Light-Emitting Applications of One-Dimensional Lead-Free Cerium(III) Metal Halides.

Combining rare earth elements with the halide perovskite structure offers valuable insights into designing nonlead (Pb) luminescent materials. However, most of these compositions tend to form zero-dimensional (0D) networks of metal-halide polyhedra, with higher-dimensional (1D, 2D, and 3D) structures receiving relatively less exploration. Herein, we present synthesis and optical properties of Cs3CeCl6·3H2O, characterized by its unique 1D crystal structure. The conduction band minimum of Cs3CeCl6·3H2O becomes less localized as a result of the increased structural dimension, making it possible for the materials to achieve an efficient electrical injection. For both Cs3CeCl6·3H2O single crystals and nanocrystals, we also observed remarkable luminescence with near-unity photoluminescence quantum yield and exceptional phase stability. Cs3CeCl6·3H2O single crystals demonstrate an X-ray scintillation light yield of 31900 photons/MeV, higher than that of commercial LuAG:Ce (22000 photons/MeV); electrically driven light-emitting diodes fabricated with Cs3CeCl6·3H2O nanocrystals yield the characteristic emission of Ce3+, indicating their potential use in next-generation violet-light-emitting devices.

Direct chemical induction of hepatocyte-like cells with capacity for liver repopulation.

BACKGROUND AND AIMS: Cell fate can be directly reprogrammed from accessible cell types (e.g., fibroblasts) into functional cell types by exposure to small molecule stimuli. However, no chemical reprogramming method has been reported to date that successfully generates functional hepatocyte-like cells that can repopulate liver tissue, casting doubt over the feasibility of chemical reprogramming approaches to obtain desirable cell types for therapeutic applications. APPROACH AND RESULTS: Here, through chemical induction of phenotypic plasticity, we provide a proof-of-concept demonstration of the direct chemical reprogramming of mouse fibroblasts into functional hepatocyte-like cells using exposure to small molecule cocktails in culture medium to successively stimulate endogenous expression of master transcription factors associated with hepatocyte development, such as hepatocyte nuclear factor 4a, nuclear receptor subfamily 1, group I, member 2, and nuclear receptor subfamily 1, group H, member 4. RNA sequencing analysis, metabolic assays, and in vivo physiological experiments show that chemically induced hepatocytes (CiHeps) exhibit comparable activity and function to primary hepatocytes, especially in liver repopulation to rescue liver failure in fumarylacetoacetate hydrolase -/- recombination activating gene 2 -/- interleukin 2 receptor, gamma chain -/- mice in vivo . Single-cell RNA-seq further revealed that gastrointestinal-like and keratinocyte-like cells were induced along with CiHeps, resembling the activation of an intestinal program within hepatic reprogramming as described in transgenic approaches. CONCLUSIONS: Our findings show that direct chemical reprogramming can generate hepatocyte-like cells with high-quality physiological properties, providing a paradigm for establishing hepatocyte identity in fibroblasts and demonstrating the potential for chemical reprogramming in organ/tissue repair and regeneration therapies.

Ab Initio Self-Trapped Excitons.

We propose a new formalism and an effective computational framework to study self-trapped excitons (STEs) in insulators and semiconductors from first principles. Using the many-body Bethe-Salpeter equation in combination with perturbation theory, we are able to obtain the mode- and momentum-resolved exciton-phonon coupling matrix element in a perturbative scheme and explicitly solve the real space localization of the electron (hole), as well as the lattice distortion. Further, this method allows us to compute the STE potential energy surface and evaluate the STE formation energy and Stokes shift. We demonstrate our approach using two-dimensional magnetic semiconductor chromium trihalides and a wide-gap insulator BeO, the latter of which features dark excitons, and make predictions of their Stokes shift and coherent phonon generation which we hope will spark future experiments such as photoluminescence and transient absorption studies.

Golgi Protein 73 Promotes LPS-Induced Cardiac Dysfunction via Mediating Myocardial Apoptosis and Autophagy.

ABSTRACT: Sepsis-induced cardiac dysfunction represents a major cause of high mortality in intensive care units with limited therapeutic options. Golgi protein 73 (GP73) has been implicated in various diseases. However, the role of GP73 in lipopolysaccharide (LPS)-induced cardiac dysfunction is unclear. In this study, we established a sepsis-induced cardiac dysfunction model by LPS administration in wild-type and GP73 knockout ( GP73-/- ) mice. We found that GP73 was increased in LPS-treated mouse hearts and LPS-cultured neonatal rat cardiomyocytes (NRCMs). Knockout of GP73 alleviated myocardial injury and improved cardiac dysfunction. Moreover, depletion of GP73 in NRCMs relieved LPS-induced cardiomyocyte apoptosis and activated myocardial autophagy. Therefore, GP73 is a negative regulator in LPS-induced cardiac dysfunction by promoting cardiomyocyte apoptosis and inhibiting cardiomyocyte autophagy.

Full-length circRNA sequencing method using low-input RNAs and profiling of circRNAs in MPTP-PD mice on a nanopore platform.

Considering the importance of accurate information of full-length (FL) transcripts in functional analysis, researchers prefer to develop new sequencing methods based on third-generation sequencing (TGS) rather than short-read sequencing. Several FL circRNA sequencing strategies have been developed. However, the current methods are inapplicable to low-biomass samples, since a large amount of total RNAs are acquired for circRNA enrichment before library preparation. In this work, we developed an effective method to detect FL circRNAs from a nanogram level (1-100 ng) of total RNAs based on a nanopore platform. Additionally, prior to the library preparation process, we added a series of 24 nt barcodes for each sample to reduce the cost and operating time. Using this method, we profiled circRNA expression in the striatum, hippocampus and cerebral cortex of a Parkinson's disease (PD) mouse model. Over 6% of reads were effective for FL circRNA identification in most datasets. Notably, a reduction in the RNA initial input resulted in a lower correlation between replicates and the detection efficiency for longer circRNA, but the lowest input (1 ng) was able to detect numerous FL circRNAs. Next, we systematically identified over 263 934 circRNAs in PD and healthy mice using the lower-input FL sequencing method, some of which came from 50.52% of PD-associated genes. Moreover, significant changes were observed in the circRNA expression pattern at an isoform level, and high-confidence protein translation evidence was predicted. Overall, we developed an effective method to characterize FL circRNAs from low-input samples and provide a comprehensive insight into the biological function of circRNAs in PD at an isoform level.

Organoboron flank-substituted donor-acceptor polymer anode with ultra-long cycling stability for lithium ion batteries.

The tunable structure and other properties of organic materials suggest that they can potentially solve the shortcomings of traditional anodes such as graphite. We successfully introduced an organoboron unit into the thiophene-based polymer PBT-2 to construct a donor-acceptor polymer anode. The charge delocalization and LUMO energy level resulting from the unique structure of this material enabled good redox activity and a very stable electrochemical performance in electrochemical tests, with a reversible capacity of 262 mA h g-1 at 0.5 A g-1 and >10 000 cycles at 1 A g-1 with a decay of 0.056‰ per cycle. Accordingly, targeted structural design to overcome the shortcomings of active units such as thiophene can effectively regulate their electrochemical performance, providing a solution for the development of high-performance anode materials for use in lithium ion batteries.

Organic-Inorganic Hybrid Cuprous-Based Metal Halides with Unique Two-Dimensional Crystal Structure for White Light-Emitting Diodes.

Ternary cuprous (Cu+)-based metal halides, represented by cesium copper iodide (e.g., CsCu2I3 and Cs3Cu2I5), are garnering increasing interest for light-emitting applications owing to their intrinsically high photoluminescence quantum yield and direct bandgap. Toward electrically driven light-emitting diodes (LEDs), it is highly desirable for the light emitters to have a high structural dimensionality as it may favor efficient electrical injection. However, unlike lead-based halide perovskites whose light-emitting units can be facilely arranged in three-dimensional (3D) ways, to date, nearly all ternary Cu+-based metal halides crystallize into 0D or 1D networks of Cu-X (X = Cl, Br, I) polyhedra, whereas 3D and even 2D structures remain mostly uncharted. Here, by employing a fluorinated organic cation, we report a new kind of ternary Cu+-based metal halides, (DFPD)CuX2 (DFPD+ = 4,4-difluoropiperidinium), which exhibits unique 2D layered crystal structure. Theoretical calculations reveal a highly dispersive conduction band of (DFPD)CuBr2, which is beneficial for charge carrier injection. It is also of particular significance to find that the 2D (DFPD)CuBr2 crystals show appealing properties, including improved ambient stability and an efficient warm white-light emission, making it a promising candidate for single-component lighting and display applications.

Remnant Cholesterol and Common Carotid Artery Intima-Media Thickness in Community Population with Normal Low-Density Lipoprotein Cholesterol.

INTRODUCTION: Remnant cholesterol is a risk factor for cardiovascular disease, especially when low-density lipoprotein cholesterol (LDL-C) levels are normal. However, there are few studies on the relationship between remnant cholesterol and subclinical atherosclerosis. Common carotid artery intima-media thickness (cIMT) is an imaging marker of subclinical atherosclerosis. This study aimed to investigate the relationship between remnant cholesterol and cIMT in a community population with normal LDL-C. METHODS: This study is a retrospective analysis; 1,101 community population with available carotid artery imaging and fasting lipid data with LDL-C <4.1 mmol/L were included in this analysis. Remnant cholesterol was calculated as total cholesterol minus LDL-C minus high-density lipoprotein cholesterol. Abnormal cIMT was defined as maximum cIMT value ≥1 mm. Logistic regression was used to assess the relationships between remnant cholesterol levels and abnormal cIMT. RESULTS: As the remnant cholesterol level increased from the lowest to the highest quartile, the rate of abnormal cIMT increased from 24.5% to 38.6% (p trend <0.001) in the community population with normal LDL-C level. In the unadjusted model, the odds ratios (ORs, 95% confidence intervals) in the highest quartile group were 1.937 (1.338-2.803) for abnormal cIMT compared with the lowest quartile. The multivariable-adjusted ORs (95% confidence intervals) for the highest versus lowest quartile of remnant cholesterol were 2.132 (1.420-3.202) for abnormal cIMT. CONCLUSION: Elevated fasting remnant cholesterol levels were positively associated with abnormal cIMT in community population with normal LDL-C levels. Remnant cholesterol may be an important indicator of risk stratification in community population with normal LDL-C level.

A comprehensive analysis of library preparation methods shows high heterogeneity of extrachromosomal circular DNA but distinct chromosomal amount levels reflecting different cell states.

Extrachromosomal circular DNA (eccDNA) was discovered several decades ago, but little is known about its function. With the development of sequencing technology, several library preparation methods have been developed to elucidate the biogenesis and function of eccDNA. However, different treatment methods have certain biases that can lead to their erroneous interpretation. To address these issues, we compared the performance of different library preparation methods. Our investigation revealed that the utilization of rolling-circle amplification (RCA) and restriction enzyme linearization of mitochondrial DNA (mtDNA) significantly enhanced the efficiency of enriching extrachromosomal circular DNA (eccDNA). However, it also introduced certain biases, such as an unclear peak in ∼160-200 bp periodicity and the absence of a typical motif pattern. Furthermore, given that RCA can lead to a disproportionate change in copy numbers, eccDNA quantification using split and discordant reads should be avoided. Analysis of the genomic and elements distribution of the overall population of eccDNA molecules revealed a high correlation between the replicates, and provided a possible stability signature for eccDNA, which could potentially reflect different cell lines or cell states. However, we found only a few eccDNA with identical junction sites in each replicate, showing a high degree of heterogeneity of eccDNA. The emergence of different motif patterns flanking junctional sites in eccDNAs of varying sizes suggests the involvement of multiple potential mechanisms in eccDNA generation. This study comprehensively compares and discusses various essential approaches for eccDNA library preparation, offering valuable insights and practical advice to researchers involved in characterizing eccDNA.

Optical system design of a DMD-SHS combined modulation interference spectrometer.

Digital micromirror device (DMD) and spatial heterodyne spectroscopy (SHS) combined modulation interference spectroscopy (DMD-SHS) introduces a DMD for the secondary modulation of interferometric data to achieve a Hadamard transform. DMD-SHS can improve the performance index of the spectrometer in terms of the SNR, dynamic range, and spectral bandwidth, while retaining the advantages of a conventional SHS. The DMD-SHS optical system is more complex than a traditional SHS, which places more demands on the optical system's spatial layout and the optical components' performance. According to the DMD-SHS modulation mechanism, the functions of the main components were analyzed, and their design requirements were determined. Based on the potassium spectra detection, a DMD-SHS experimental device was designed. The potassium lamp and integrating sphere detection experiments demonstrated the detection capability of the DMD-SHS experimental device with a spectral resolution of 0.0327 nm and a spectral range of 763.66∼771.25n m, which thoroughly verified the feasibility of DMD and SHS combined modulation interference spectroscopy.

Cell-Free DNA Fragmentomics: The Novel Promising Biomarker.

Cell-free DNA molecules are released into the plasma via apoptotic or necrotic events and active release mechanisms, which carry the genetic and epigenetic information of its origin tissues. However, cfDNA is the mixture of various cell fragments, and the efficient enrichment of cfDNA fragments with diagnostic value remains a great challenge for application in the clinical setting. Evidence from recent years shows that cfDNA fragmentomics' characteristics differ in normal and diseased individuals without the need to distinguish the source of the cfDNA fragments, which makes it a promising novel biomarker. Moreover, cfDNA fragmentomics can identify tissue origins by inferring epigenetic information. Thus, further insights into the fragmentomics of plasma cfDNA shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In this review, we focus on the cfDNA fragment characteristics and its potential application, such as fragment length, end motifs, jagged ends, preferred end coordinates, as well as nucleosome footprints, open chromatin region, and gene expression inferred by the cfDNA fragmentation pattern across the genome. Furthermore, we summarize the methods for deducing the tissue of origin by cfDNA fragmentomics.

Spatial Transcriptome Profiling of Mouse Hippocampal Single Cell Microzone in Parkinson's Disease.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and cognitive functions. It is still unknown how gene expression changes in single-cell in different spatial locations of the hippocampus of Parkinson's disease. The purpose of this study was to analyze the gene expression features of single cells in different spatial locations of mouse hippocampus, and to explore the effects of gene expression regulation on learning and memory mechanisms. Here, we obtained 74 single-cell samples from different spatial locations in a mouse hippocampus through microdissection technology, and used single-cell RNA-sequencing and spatial transcriptome sequencing to visualize and quantify the single-cell transcriptome features of tissue sections. The results of differential expression analysis showed that the expression of Sv2b, Neurod6, Grp and Stk32b genes in a hippocampus single cell at different locations was significantly different, and the marker genes of CA1, CA3 and DG subregions were identified. The results of gene function enrichment analysis showed that the up-regulated differentially expressed genes Tubb2a, Eno1, Atp2b1, Plk2, Map4, Pex5l, Fibcd1 and Pdzd2 were mainly involved in neuron to neuron synapse, vesicle-mediated transport in synapse, calcium signaling pathway and neurodegenerative disease pathways, thus affecting learning and memory function. It revealed the transcriptome profile and heterogeneity of spatially located cells in the hippocampus of PD for the first time, and demonstrated that the impaired learning and memory ability of PD was affected by the synergistic effect of CA1 and CA3 subregions neuron genes. These results are crucial for understanding the pathological mechanism of the Parkinson's disease and making precise treatment plans.

Two-Step Redox in Polyimide: Witness by In Situ Electron Paramagnetic Resonance in Lithium-ion Batteries.

Organic materials are promising candidates for future rechargeable batteries, owing to their high natural abundance and rapidly redox reaction. Elaborating the charge/discharge process of organic electrode is critical to unveil the fundamental redox mechanism of lithium-ion batteries (LIBs), but monitoring of this process is still challenging. Here, we report a nondestructive electron paramagnetic resonance (EPR) technique to real-time detect the electron migration step within polyimide cathode. From in situ EPR tests, we vividly observe a classical redox reaction along with two-electron transfer which only shows one pair of peaks in the cyclic voltammetry curve. The radical anion and dianion intermediates are detailed delineation at redox sites in EPR spectra, which can be further confirmed through density functional theory calculations. This approach is especially crucial to elaborate the correlation behind electrochemical and molecular structure for multistep organic-based LIBs.

Novel Method of Full-Length RNA-seq That Expands the Identification of Non-Polyadenylated RNAs Using Nanopore Sequencing.

The occurrence of diseases displayed transcriptome alteration, including both coding and non-coding transcripts. The third-generation sequencing (TGS) technologies allow for intensive and comprehensive research of the transcriptome. However, the present standard TGS RNA sequencing method is unable to detect many of the non-polyadenylated [non-poly(A)] RNAs. To obtain more complete transcriptome information, we presented a new comprehensive sequencing approach by performing conventional poly(A) RNA-sequencing combined with the sequencing of non-poly(A) RNA fraction which was tailed by poly(U) on HepG2 and HL-7702 cell lines, enabling the detection of multiple categories of non-poly(A) RNAs excluded by the existing standard approach. Moreover, the length distribution of the full-splice match transcripts was longer than that assembled by short-reads, which contributed to characterizing alternative splicing events and provided a comprehensive portrait of transcriptional complexity. Besides the detection of genes with differential expression patterns in the poly(A) library between HepG2 and HL-7702, we also found a cancer-related non-coding gene in the poly(U) data, that is, growth arrest special 5 (GAS5). Collectively, our results suggested that the novel method effectively captured both poly(A) and non-poly(A) transcripts in the tested cell lines and allowed a deeper exploration of the transcriptome.

One Stone Two Birds: Unlocking the Synergy between Amorphous Ni(OH)2 and Pd Nanocrystals toward Ethanol and Formic Acid Oxidation.

Even though extensive efforts have been devoted to mixing Pd nanocrystals with Ni(OH)2 for the enhanced synergy, it remains a great challenge to incorporate nanosized Ni(OH)2 species on the Pd electrode and reveal their synergy. Herein, we present spongelike Pd nanocrystals with the modification of amorphous Ni(OH)2 species. The catalyst configuration is first considered by compositing Pd with Ni(OH)2 species to optimize the Pd-Pd interatomic distance and then constructing a strongly coupled interface between Pd nanostructures and Ni(OH)2 species. For the ethanol oxidation reaction (EOR) and the formic acid oxidation reaction (FAOR), Pd-Ni(OH)2 composites exhibit an impressive mass activity of 4.98 and 2.65 A mgPd-1, respectively. Most impressively, there is no significant decrease in the EOR activity during five consecutive cycles (50 000 s). A series of CO-poisoning tests have proved that the enhanced EOR and FAOR performances involve synergy between Pd nanostructures and Ni(OH)2 species.

Comparative analysis of circular RNA enrichment methods.

The circRNAs sequencing results vary due to the different enrichment methods and their performance is needed to systematic comparison. This study investigated the effects of different circRNA enrichment methods on sequencing results, including abundance and species of circRNAs, as well as the sensitivity and precision. This experiment was carried out by following four common circRNA enrichment methods: including ribosomal RNA depletion (rRNA-), polyadenylation and poly (A+) RNA depletion followed by RNase R treatment (polyA+RNase R), rRNA-+polyA+RNase R and polyA+RNase R+ rRNA-. The results showed that polyA+RNase R+ rRNA - enrichment method obtained more circRNA number, higher sensitivity and abundance among them; polyA+RNase R method obtained higher precision. The linear RNAs can be thoroughly removed in all enrichment methods except rRNA depletion method. Overall, our results helps researchers to quickly selection a circRNA enrichment of suitable for own study among many enrichment methods, and it provides a benchmark framework for future improvements circRNA enrichment methods.[Figure: see text].