scholarly journals High-resolution, strand-specific R-loop mapping via S9.6-based DNA–RNA immunoprecipitation and high-throughput sequencing

2019 ◽  
Vol 14 (6) ◽  
pp. 1734-1755 ◽  
Author(s):  
Lionel A. Sanz ◽  
Frédéric Chédin
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Immunoprecipitation

scholarly journals High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes

Life ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 58
Author(s):  
Aida Martinez-Sanchez ◽  
Stefano Lazzarano ◽  
Eshita Sharma ◽  
Helen Lockstone ◽  
Christopher L. Murphy
Keyword(s):  
Cartilage Repair ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Articular Chondrocytes ◽  
Articular Chondrocyte ◽  
Coding Region ◽  
Cartilage Development ◽  
Rna Immunoprecipitation ◽  
Important Regulator ◽  
Mmp13 Expression

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3′UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

scholarly journals Episo: quantitative estimation of RNA 5-methylcytosine at isoform level by high-throughput sequencing of RNA treated with bisulfite

2019 ◽  
Vol 36 (7) ◽  
pp. 2033-2039 ◽  
Author(s):  
Junfeng Liu ◽  
Ziyang An ◽  
Jianjun Luo ◽  
Jing Li ◽  
Feifei Li ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Quantitative Estimation ◽  
Supplementary Information ◽  
Biological Processes ◽  
Single Nucleotide ◽  
Rna Immunoprecipitation ◽  
Nucleotide Resolution ◽  
Human And Mouse ◽  
Single Nucleotide Resolution

Abstract Motivation RNA 5-methylcytosine (m5C) is a type of post-transcriptional modification that may be involved in numerous biological processes and tumorigenesis. RNA m5C can be profiled at single-nucleotide resolution by high-throughput sequencing of RNA treated with bisulfite (RNA-BisSeq). However, the exploration of transcriptome-wide profile and potential function of m5C in splicing remains to be elucidated due to lack of isoform level m5C quantification tool. Results We developed a computational package to quantify Epitranscriptomal RNA m5C at the transcript isoform level (named Episo). Episo consists of three tools: mapper, quant and Bisulfitefq, for mapping, quantifying and simulating RNA-BisSeq data, respectively. The high accuracy of Episo was validated using an improved m5C-specific methylated RNA immunoprecipitation (meRIP) protocol, as well as a set of in silico experiments. By applying Episo to public human and mouse RNA-BisSeq data, we found that the RNA m5C is not evenly distributed among the transcript isoforms, implying the m5C may subject to be regulated at isoform level. Availability and implementation Episo is released under the GNU GPLv3+ license. The resource code Episo is freely accessible from https://github.com/liujunfengtop/Episo (with Tophat/cufflink) and https://github.com/liujunfengtop/Episo/tree/master/Episo_Kallisto (with Kallisto). Supplementary information Supplementary data are available at Bioinformatics online.

HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing

2013 ◽  
Vol 83 (1) ◽  
pp. 10-16 ◽  
Author(s):  
Y. Ozaki ◽  
S. Suzuki ◽  
A. Shigenari ◽  
Y. Okudaira ◽  
E. Kikkawa ◽  
...  
Keyword(s):  
High Resolution ◽  
Long Range ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Resolution Level ◽  
Long Range Pcr

P–556 NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Xiang ◽  
C Zhou ◽  
Q Guo ◽  
X Liang
Keyword(s):  
Mass Spectrometry ◽  
Oocyte Maturation ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Regulatory Role ◽  
Maturation In Vitro ◽  
Rna Immunoprecipitation ◽  
Transcriptional Modulation

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P < 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings: Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number Not applicable

P-556 Pre-selected for an award: NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Xiang ◽  
C Zhou ◽  
Q Guo ◽  
X Liang
Keyword(s):  
Mass Spectrometry ◽  
Oocyte Maturation ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Regulatory Role ◽  
Maturation In Vitro ◽  
Rna Immunoprecipitation ◽  
Transcriptional Modulation

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P < 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number not applicable

scholarly journals HTSSIP: an R package for analysis of high throughput sequencing data from nucleic acid stable isotope probing (SIP) experiments

2017 ◽  
Author(s):  
Nicholas D. Youngblut ◽  
Samuel E. Barnett ◽  
Daniel H. Buckley
Keyword(s):  
High Resolution ◽  
Stable Isotope ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
R Package ◽  
Stable Isotope Probing ◽  
Sequencing Data ◽  
Metabolic Processes ◽  
Link Type ◽  
High Throughput Sequencing Data

AbstractCombining high throughput sequencing with stable isotope probing (HTS-SIP) is a powerful method for mapping in situ metabolic processes to thousands of microbial taxa. However, accurately mapping metabolic processes to taxa is complex and challenging. Multiple HTS-SIP data analysis methods have been developed, including high-resolution stable isotope probing (HR-SIP), multi-window high-resolution stable isotope probing (MW-HR-SIP), quantitative stable isotope probing (q-SIP), and ΔBD. Currently, the computational tools to perform these analyses are either not publicly available or lack documentation, testing, and developer support. To address this shortfall, we have developed the HTSSIP R package, a toolset for conducting HTS-SIP analyses in a straightforward and easily reproducible manner. The HTSSIP package, along with full documentation and examples, is available from CRAN at https://cran.r-project.org/web/packages/HTSSIP/index.html and Github at https://github.com/nick-youngblut/HTSSIP.

scholarly journals Chromosome Conformation Capture with Nuclear Titrated Capture-C (NuTi Capture-C)

2021 ◽  
Author(s):  
Damien J. Downes ◽  
Jim R. Hughes
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Chromosome Conformation Capture ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Chromosome Conformation ◽  
Chromatin Interactions

Abstract NuTi Capture-C is a Chromosome Conformation Capture (3C) approach, which can very efficiently identify chromatin interactions at target viewpoints at high resolution. The addition of high-throughput sequencing adaptors prior to enrichment allows for multiplexing of replicates and comparison samples. This method is an improvement on the previous NG Capture-C1 method in that modifications have been made to the in situ 3C method to improve nuclear integrity (Nuclear 3C). Additionally, capture has been optimised to viewpoint complexity through titration, maximising on target sequence specificity. The experiment will take several weeks and provide reproducible interaction profiles for tens to thousands of viewpoints of interest.

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