MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cellsXiaonan Hu - 2022
AbstractPurpose
Transforming growth factor-beta (TGFB)-mediated epithelial-mesenchymal transition (EMT) plays a crucial role in the pathogenesis of retinal fibrosis, which is one of the leading causes of impaired vision. Current approaches to treating retinal fibrosis focus, among other things, on inhibiting the TGFB signaling pathway. Transient expression of microRNAs (miRNAs) is one way to inhibit the TGFB pathway post-transcriptionally. Our previous study identified the miRNA miR-302d as a regulator of multiple TGFB-related genes in ARPE-19 cells. To further explore its effect on primary cells, the effect of miR-302d on TGFB-induced EMT in primary human retinal pigment epithelium (hRPE) was investigated in vitro.
Methods
hRPE cells were extracted from patients receiving enucleation. Transfection of hRPE cells with miR-302d was performed before or after TGFB1 stimulation. Live-cell imaging, immunocytochemistry staining, Western blot, and ELISA assays were utilized to identify the alterations of cellular morphology and EMT-related factors expressions in hRPE cells.
Results
hRPE cells underwent EMT by TGFB1 exposure. The transfection of miR-302d inhibited the transition with decreased production of mesenchymal markers and increased epithelial factors. Meanwhile, the phosphorylation of SMAD2 activated by TGFB1 was suppressed. Moreover, miR-302d expression promoted TGFB1-induced fibroblast-like hRPE cells to revert towards an epithelial stage. As confirmed by ELISA, miR-302d reduced TGFB receptor 2 (TGFBR2) and vascular endothelial growth factor A (VEGFA) levels 48 hours after transfection.
Conclusions
The protective effect of miR-302d might be a promising approach for ameliorating retinal fibrosis and neovascularization. MiR-302d suppresses TGFB-induced EMT in hRPE cells via downregulation of TGFBR2, even reversing the process. Furthermore, miR-302d reduces the constitutive secretion of VEGFA from hRPE cells.
Stress deficits in reward behaviour are associated with and replicated by dysregulated amygdala-nucleus accumbens pathway functionLorraine Madur - 2022
AbstractReduced reward interest/learning and reward-to-effort valuation are distinct, common symptoms in
neuropsychiatric disorders for which chronic stress is a major aetiological factor. Glutamate neurons in
basal amygdala (BA) project to various regions including nucleus accumbens (NAc). The BA-NAc neural
pathway is activated by reward and aversion, with many neurons being monovalent. In adult male mice,
chronic social stress (CSS) led to both reduced discriminative reward learning (DRL) associated with
decreased BA-NAc activity, and reduced reward-to-effort valuation (REV) associated, in contrast, with
increased BA-NAc activity. Chronic tetanus toxin BA-NAc inhibition replicated the CSS-DRL effect and
caused a mild REV reduction, whilst chronic DREADDs BA-NAc activation replicated the CSS effect on
REV without affecting DRL. This study provides novel evidence that stress disruption of reward
processing involves the BA-NAc neural pathway; the bi-directional effects implicate opposite activity
changes in reward (learning) neurons and aversion (effort) neurons in the BA-NAc pathway following
chronic stress.
DETERMINATION OF PROTEIN LOCALIZATION AND RNA KINETICS IN HUMAN CELLSRoberto Arsie - 2022
AbstractThe intracellular distribution of a specific protein is essential for its function. Proper protein localization is therefore an essential biological process, regulated at many levels and central for eukaryotic compartments organization. Consequently, several methods have been established to globally profile proteomes at different subcellular regions. Many of these approaches rely on the different biochemical features of eukaryotic compartments to separate them and analyse their relative protein composition in parallel. In this study we assessed the protein distribution of HEK293 cells using BirA* proximity labelling activity and restricting its localization at cellular regions difficult to purified with traditional methods: the cytosol-facing sides of the endoplasmic reticulum, mitochondria, and plasma membranes. We obtained high-quality datasets for each of the compartment examined, both recapitulating the protein distribution known from literature and discovering new candidates at the investigated subcellular regions. Interestingly, we also identified proteins localizing at more than one subcellular compartment, providing useful insights on the protein composition shared by different organelles. Proteins with different biological activity were captured by our proximity labelling approach, such as metabolic enzymes and integral receptors. RNA-binding proteins (RBPs) were also found in our datasets, but the direct interactions between them and RNA molecules could not be validated using only BirA* biotinylation. To obtain an activity-based RBP profiling and assess the composition of protein binding to RNA in different cellular compartments, we developed the f-XRNAX protocol. It consists of UV-crosslinking, cellular fractionation and organic-phase enrichment of RBPs coupled with mass spectrometry. We recovered background-corrected proteomes for nuclei, cytoplasm and membranes of HEK293 cells. Protein domain analysis of peptides crosslinked to ribonucleotides showed an enrichment in known RNA-binding domains. We also determined the protein domains where the RNA-protein contacts occurred in vivo using enriched peptides profiles and reported some cases of differential domain usage of the same protein in two different compartments. Surprisingly, many non-canonical RBPs were identified in the membrane fraction, and their peptide profiles were enriched in regions with high density of intrinsically disordered regions, indicating a possibly weak interaction with RNA mediated by these non-structural motives. Lastly, we provided evidence of the differential binding to RNA of the same protein in different HEK293 compartments. In the second part of this thesis, we focused on the determination and quantification of newly transcribed RNAs at the single-cell level. The kinetics of RNA transcription, processing and degradation were until recently not measurable at the single-cell level. Thus, we have developed a novel approach (called SLAM-Drop-seq ) by adapting the published SLAM-seq method to single cells. We used SLAMDrop-seq to estimate time-dependent RNA kinetics rates of transcription and turnover for hundreds ZUSAMMENFASSUNG 3 of oscillating transcripts during the cell cycle of HEK293 cells. We found that genes regulate their expression with different strategies and have specific modes to fine-tune their kinetic rates along the cell cycle.
The reference genome of the Vernal Pool Tadpole Shrimp, Lepidurus packardiShannon Rose Kieran Blair - 2022
AbstractIn this paper we report on the scaffold-level assembled genome for the federally endangered, California endemic crustacean Lepidurus packardi (the Vernal Pool Tadpole Shrimp). Lepidurus packardi is a key food source for other conserved California species including the California Tiger Salamander Ambystoma californiense. It faces significant habitat loss and fragmentation as vernal pools are threatened by urbanization, agricultural conversion and climate change. This resource represents the first scaffold-level genome of any Lepidurus species. The assembled genome spans 108.6 Mbps, with six chromosome-length scaffolds comprising 71% of total genomic length and 444 total contigs. The BUSCO score for this genome is 97.3%, suggesting a high level of completeness. We produced a predicted gene set for this species trained on the Daphnia magna set of genes and predicted 17,650 genes. These tools can aid researchers in understanding the evolution and adaptive potential of alternative reproductive modes within this species.
Plasma contains ultrashort single-stranded DNA in addition to nucleosomal cell-free DNAJordan Cheng - 2022
AbstractPlasma cell-free DNA is being widely explored as a biomarker for clinical screening. Currently, methods are optimized for the extraction and detection of double-stranded mononucleosomal cell-free DNA of ∼160bp in length. We introduce uscfDNA-seq, a single-stranded cell-free DNA next-generation sequencing pipeline, which bypasses previous limitations to reveal a population of ultrashort single-stranded cell-free DNA in human plasma. This species has a modal size of 50nt and is distinctly separate from mononucleosomal cell-free DNA. Treatment with single-stranded and double-stranded specific nucleases suggests that ultrashort cell-free DNA is primarily single-stranded. It is distributed evenly across chromosomes and has a similar distribution profile over functional elements as the genome, albeit with an enrichment over promoters, exons, and introns, which may be suggestive of a terminal state of genome degradation. The examination of this cfDNA species could reveal new features of cell death pathways or it can be used for cell-free DNA biomarker discovery.
Neuron-specific translational control shift ensures proteostatic resilience during ER stressKimberly Wolzak - 2022
AbstractProteostasis is essential for cellular survival and particularly important for highly specialised post-mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R-like endoplasmic reticulum (ER) kinase (PERK)-mediated phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type-specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK-deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive
translational control in PERK-deficient neurons. Haem-regulated inhibitor (HRI) mediates p-eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back-up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress.
Characterization of the detrimental effects of type IV glandular trichomes on the aphid Macrosiphum euphorbiae in tomatoLidia Blanco-Sánchez, - 2021
AbstractBACKGROUND: Glandular trichomes are essential in plants' defence against pests however, the mechanisms of action are not
completely understood. While there is considerable evidence of feeding and movement impairment by trichomes, the
effect on other traits is less clear. We combined laboratory and greenhouse experiments with molecular analysis to understand how glandular trichomes affect the behavior, population growth, and the expression of biomarkers involved in
detoxification, primary metabolism, and developmental pathways of the aphid Macrosiphum euphorbiae. We used two
isogenic tomato lines that differ in the presence of type IV glandular trichomes and production of acylsucroses;
i.e.,Solanum lycopersicum cv. ‘Moneymaker’ and an introgressed line from Solanum pimpinellifolium (with trichomes type
IV).
RESULTS: Type IV glandular trichomes affected host selection and aphid proliferation with aphids avoiding, and showing
impaired multiplication on the genotype with trichomes. The exposure to type IV glandular trichomes resulted in the overexpression of detoxication markers (i.e., Hsp70, Hsp17, Hsp10); the repression of the energetic metabolism (GAPDH), and the activation of the ecdysone pathway; all these, underlying the key adaptations and metabolic trade-offs in aphids exposed to
glandular trichomes.
CONCLUSION: Our results demonstrate the detrimental effect of glandular trichomes (type IV) on the aphid and put forward
their mode of action. Given the prevalence of glandular trichomes in wild and cultivated Solanaceae; and of the investigated
molecular biomarkers in insects in general, our results provide relevant mechanisms to understand the effect of trichomes
not only on herbivorous insects but also on other trophic levels.
Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis InfectionSara F. Martins Gomes - 2019
AbstractMeningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.
An unconventional RNA-based thermosensor within the 5’ UTR of Staphylococcus aureus cidAHebaallaha Hussein - 2019
AbstractStaphylococcus aureus is a Gram-positive bacterial pathogen of global concern and a leading cause of bacterial infections worldwide. Asymptomatic carriage of S. aureus on the skin and in the anterior nares is common and recognized as a predisposing factor to invasive infection. Transition of S. aureus from the carriage state to that of invasive infection is often accompanied by a temperature upshift from approximately 33°C to 37°C. Such a temperature shift is known in other pathogens to influence gene expression, often resulting in increased production of factors that promote survival or virulence within the host. One mechanism by which bacteria modulate gene expression in response to temperature is by the regulatory activity of RNA-based thermosensors, cis-acting riboregulators that control translation efficiency. This study was designed to identify and characterize RNA-based thermosensors in S. aureus. Initially predicted by in silico analyses of the S. aureus USA300 genome, reporter-based gene expression analyses and site-specific mutagenesis were performed to demonstrate the presence of a functional thermosensor within the 5’ UTR of cidA, a gene implicated in biofilm formation and survival of the pathogen. The nucleic sequence composing the identified thermosensor are sufficient to confer temperature-dependent post-transcriptional regulation, and activity is predictably altered by the introduction of site-specific mutations designed to stabilize or destabilize the structure within the identified thermosensor. The identified regulator is functional in both the native bacterial host S. aureus and in the distally related species Escherichia coli, suggesting that its regulatory activity is independent of host-specific factors. Interestingly, unlike the majority of bacterial RNA-based thermosensors characterized to date, the cidA thermosensor facilitates increased target gene expression at lower temperatures. In addition to the characterization of the first RNA-based thermosensor in the significant pathogen S. aureus, it highlights the diversity of function within this important class of ribo-regulators.
Symbiotic bacteria associated with ascidian vanadium accumulation identified by 16S rRNA amplicon sequencingTatsuya Ueki - 2019
AbstractAscidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life.
HCN4 knockdown in dorsal hippocampus promotes anxiety‐like behavior in miceAnne Gunther - 2019
AbstractHyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channels mediate the Ih current in the murine hippocampus. Disruption of the Ih current by knockout of HCN1, HCN2 or tetratricopeptide repeat‐containing Rab8b‐interacting protein has been shown to affect physiological processes such as synaptic integration and maintenance of resting membrane potentials as well as several behaviors in mice, including depressive‐like and anxiety‐like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus‐based behaviors in mice. We show that HCN4 is expressed in various regions of the hippocampus, with distinct expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno‐associated viral (rAAV) vectors mediating expression of short hairpin RNA against hcn4 (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was evident in mice treated with shHcn4 compared to control littermates. Our findings suggest that HCN4 specifically contributes to anxiety‐like behaviors in mice.
Expression of ELF1, a lymphoid ETS domain‐containing transcription factor, is recurrently lost in classical Hodgkin lymphomaJulia Paczkowska - 2019
AbstractLoss of B cell-specific transcription factors (TFs) and the resulting loss ofB-cell phenotype of Hodgkin and Reed-Sternberg (HRS) cells is a hallmarkof classical Hodgkin lymphoma (cHL). Here we have analysed two mem-bers of ETS domain containing TFs, ELF1 and ELF2,regarding (epi)ge-nomic changes as well as gene and protein expression. We observedabsence or lower levels of ELF1 protein in HRS cells of 31/35 (89%) casescompared to the bystander cells and significant (P<001) downregulationof the gene on mRNA as well as protein level in cHL compared to non-cHL cell lines. However, no recurrent loss of ELF2 protein was observed.Moreover,ELF1was targeted by heterozygous deletions combined withhypermethylation of the remaining allele(s) in 4/7 (57%) cell lines. Indeed,DNA hypermethylation (range 95–99%, mean 98%) detected in the vicinityof theELF1transcription start site was found in all 7/7 (100%) cHL celllines. Similarly, 5/18 (28%) analysed primary biopsies carried heterozygousdeletions of the gene. We demonstrate that expression of ELF1 is impairedin cHL through genetic and epigenetic alterations, and thus, it may repre-sent an additional member of a TF network whose downregulation con-tributes to the loss of B-cell phenotype of HRS cells.
https://academic.oup.com/hmg/advance-article-abstract/doi/10.1093/hmg/ddy292/5068613?redirectedFrom=fulltextFeiran Zhang - 2018
AbstractN6-methyladenosine (m6A) is the most prevalent internal modification of mammalian messenger RNAs (mRNAs) and long non-coding RNAs. The biological functions of this reversible RNA modification can be interpreted by cytoplasmic and nuclear “m6A reader” proteins to fine-tune gene expression, such as mRNA degradation and translation initiation. Here we profiled transcriptome-wide m6A sites in adult mouse cerebral cortex, underscoring that m6A is a widespread epitranscriptomic modification in brain. Interestingly, the mRNA targets of fragile X mental retardation protein (FMRP), a selective RNA-binding protein, are enriched for m6A marks. Loss of functional FMRP leads to Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Transcriptome-wide gene expression profiling identified 2,035 genes differentially expressed in the absence of FMRP in cortex, and 92.5% of 174 downregulated FMRP targets are marked by m6A. Biochemical analyses indicate that FMRP binds to the m6A sites of its mRNA targets and interacts with m6A reader YTHDF2 in an RNA-independent manner. FMRP maintains the stability of its mRNA targets while YTHDF2 promotes the degradation of these mRNAs. These data together suggest that FMRP regulates the stability of its m6A-marked mRNA targets through YTHDF2, which could potentially contribute to the molecular pathogenesis of FXS.
Transcriptome of Xenopus andrei, an octoploid frog, during embryonic developmentMark E. Pownall - 2018
AbstractAlthough polyploidy occurs throughout the fish and amphibian lineages, the Xenopus genus exhibits a high incidence of polyploidy, with 25 out of the 26 known species being polyploid. However, transcriptomic information is currently available for only one of these species, the tetraploid Xenopus laevis. Xenopus andrei, an octoploid species within the Xenopus genus, offers an opportunity for assessing a novel polyploid transcriptome during vertebrate development. RNA-Seq data was generated at nine different developmental stages ranging from unfertilized eggs through swimming tadpole stages and raw FASTQ files were deposited in the NCBI SRA database (accession number SRP134281). Additionally, RNA-seq data from all nine stages were pooled to create a de novo assembly of the transcriptome using Trinity and has been deposited in the NCBI GEO database (accession number GSE111639). To our knowledge, this represents the first published assembly of an octoploid vertebrate transcriptome. In total, 849Mb were assembled, which led to the identification of 1,650,048 transcripts in the assembly with a contig N50 of 630 bases. This RNA-Seq and transcriptome data will be valuable for comparing polyploid transcriptomes across Xenopus species, as well as understanding evolutionary implications of whole-genome duplication and polyploidy in vertebrates.
Maternal obesity during lactation may protect offspring from high fat diet-induced metabolic dysfunctionJenifer Monks - 2018
AbstractBackground/Objectives
The current obesity epidemic has spurred exploration of the developmental origin of adult heath and disease. A mother’s dietary choices and health can affect both the early wellbeing and lifelong disease-risk of the offspring.
Subjects/Methods
To determine if changes in the mother’s diet and adiposity have long-term effects on the baby’s metabolism, independently from a prenatal insult, we utilized a mouse model of diet-induced-obesity and cross-fostering. All pups were born to lean dams fed a low fat diet but were fostered onto lean or obese dams fed a high fat diet. This study design allowed us to discern the effects of a poor diet from those of mother’s adiposity and metabolism. The weaned offspring were placed on a high fat diet to test their metabolic function.
Results
In this feeding challenge, all male (but not female) offspring developed metabolic dysfunction. We saw increased weight gain in the pups nursed on an obesity-resistant dam fed a high fat diet, and increased pathogenesis including liver steatosis and adipose tissue inflammation, when compared to pups nursed on either obesity-prone dams on a high fat diet or lean dams on a low fat diet.
Conclusion
Exposure to maternal over-nutrition, through the milk, is sufficient to shape offspring health outcomes in a sex- and organ-specific manner, and milk from a mother who is obesity-prone may partially protect the offspring from the insult of a poor diet.
The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae)Óscar Herrero - 2018
AbstractBisphenol S (BPS) is an industrial alternative to the endocrine disruptor bisphenol A (BPA), and can be found in many products labeled “BPA-free”. Its use has grown in recent years, and presently it is considered a ubiquitous emerging pollutant. To date there is a lack of information on the effects of BPS on invertebrates, although they represent more than 95% of known species in the animal kingdom and are crucial for the structure and proper function of ecosystems. In this study, real-time RT-PCR was used to determine the early detrimental effects of BPS on the transcriptional rate of genes in the model species Chironomus riparius, specifically those related to the ecdysone pathway (EcR, ERR, E74, Vtg, cyp18a1) crucial for insect development and metamorphosis, stress and biotransformation mechanisms (hsp70, hsp40, cyp4g, GPx, GSTd3) that regulate adaptive responses and determine survival, and ribosome biogenesis (its2, rpL4, rpL13) which is essential for protein synthesis and homeostasis. While 24-hour exposure to 0.5, 5, 50, and 500 μg/L BPS had no effect on larval survival, almost all the studied genes were upregulated following a non-monotonic dose-response curve. Genes with the greatest increases in transcriptional activity (fold change relative to control) were EcR (3.8), ERR (2), E74 (2.4), cyp18a1 (2.5), hsp70 (1.7), hsp40 (2.5), cyp4g (6.4), GPx (1.8), and GST (2.1), while others including Vtg, GAPDH, and selected ribosomal genes remained stable. We also measured the transcriptional activity of these genes 24 hours after BPS withdrawal and a general downregulation compared to controls was observed, though not significant in most cases. Our findings showed that BPS exposure altered the transcriptional profile of these genes, which may have consequences for the hormone system and several metabolic pathways. Although further research is needed to elucidate its mode of action, these results raise new concerns about the safety of BPA alternatives.
Quantitative Bromodeoxyuridine Immunoprecipitation Analyzed by High-Throughput Sequencing (qBrdU-Seq or QBU)Joanna E. Haye-Bertolozzi - 2017
AbstractIncorporation into DNA of nucleoside analogs like 5-bromo-2′-deoxyuridine (BrdU) is a powerful tool for in vivo studies of DNA synthesis during replication and repair. Immunoprecipitation of BrdU-labeled DNA analyzed by DNA sequencing (BrdU-IP-seq) allows for genome-wide, sequence-specific tracking of replication origin and replication fork dynamics under different conditions, such as DNA damage and replication stress, and in mutant strains. We have recently developed a quantitative method for BrdU-IP-seq (qBrdU-seq) involving DNA barcoding to enable quantitative analysis of multiple experimental samples subjected to BrdU-IP-seq. After initial barcoding of multiple, individually BrdU-labeled genomic DNA samples, a pooling strategy is used for all subsequent steps including immunoprecipitation, amplification, and sequencing, which eliminates sample-to-sample variability in these steps. Parallel processing of an aliquot of the pooled input sample provides a direct control for the normalization of the data and yields results that allow quantitative comparisons of the experimental samples. Though developed for the analysis of S. cerevisiae, this method should be directly adaptable to other model systems.
