Éditeur(s) : HAL CCSD Endocrine Society
Résumé : International audience
Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.
ISSN: 0888-8809

hal-01146846
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01146846
DOI : 10.1210/me.2014-1356
PUBMED : 25710594

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
BACKGROUND: Phthalic acid esters are widely used in the manufacture of plastics. Numerous studies have shown that these phthalates impair testicular testosterone production in the rat. However, the scarce and contradictory data concerning humans have cast doubt over whether these compounds are also anti-androgenic in man. We therefore investigated the direct effects of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on organo-cultured adult human testis and a human cell line. METHODS: Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with DEHP or MEHP. The effects of ketoconazole, used as a reference molecule, were also assessed. RESULTS: In both models, DEHP and MEHP significantly inhibited testosterone production. The effects of both phthalates appeared to be specific for steroidogenesis, as INSL3 production by Leydig cells was not altered. Furthermore, the phthalates of interest had no effect on inhibin B production by Sertoli cells or on germ cell apoptosis. As only a small fraction of the phthalates added was found in the testis explants, and as these compounds were found to be metabolized, we estimate that the anti-androgenic effects observed occurred at concentrations of phthalates that are of the same order of magnitude as exposures reported in the literature for men. CONCLUSIONS: We provide the first evidence that DEHP and MEHP can inhibit testosterone production in the adult human testis. This is consistent with recent epidemiological findings of an inverse correlation between exposure to MEHP and testosterone concentrations.
ISSN: 0268-1161

hal-00876965
https://hal.archives-ouvertes.fr/hal-00876965
DOI : 10.1093/humrep/des069
PUBMED : 22402212

Éditeur(s) : HAL CCSD
Résumé : International audience
INTRODUCTION: The syndrome of combined pulmonary fibrosis and emphysema (CPFE) is characterized by imaging features consisting of the association of centrilobular and/or paraseptal emphysema and pulmonary fibrosis. Virtually all patients are smokers and thus at high risk of developing lung cancer. METHODS: This retrospective multicentre study was conducted by the Groupe d'Etudes et de Recherche sur les Maladies "Orphelines" Pulmonaires (GERM"O"P). RESULTS: A total of 47 patients presenting with lung cancer and CPFE syndrome were identified. All patients were smokers, with a mean of 47 pack-years. A pathological diagnosis of lung cancer was obtained for 38 (81%) patients. Histological type was squamous cell carcinoma in 17 (36%) patients, adenocarcinoma in 14 (30%), non-small-cell lung cancer not otherwise specified in three (6%), small-cell lung cancer in three (6%), and sarcomatoid carcinoma in one (2%). Overall, 20 of the 47 patients could not receive standard-of-care treatment for lung cancer, as per international recommendations or guidelines; this limitation was considered to be directly related to the CPFE syndrome in eight (40%) cases. CONCLUSION: Lung cancer in patients with CPFE syndrome represents a specific entity with a poor prognosis, that further represents the most characteristic and severe model of tobacco-related disease.
Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer

hal-01063912
https://hal.archives-ouvertes.fr/hal-01063912
DOI : 10.1097/JTO.0000000000000209
PUBMED : 25157769

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
The formation of ovarian follicles is a finely tuned process that takes place within a narrow time-window in rodents. Multiple factors and pathways have been proposed to contribute to the mechanisms triggering this process but the role of endocrine factors, especially estrogens, remains elusive. It is currently hypothesized that removal from the maternal hormonal environment permits follicle formation at birth. However, experimentally-induced maintenance of high 17β-estradiol (E2) levels leads to subtle, distinct, immediate effects on follicle formation and oocyte survival depending on the species and dose. In this study, we examined the immediate effects of neonatal E2 exposure from post-natal day (PND) 0 to PND2 on the whole organism and on ovarian follicle formation in rats. Measurements of plasma E2, estrone and their sulfate conjugates after E2 exposure showed that neonatal female rats rapidly acquire the capability to metabolize and clear excessive E2 levels. Concomitant modifications to the mRNA content of genes encoding selected E2 metabolism enzymes in the liver and the ovary in response to E2 exposure indicate that E2 may modify the neonatal maturation of these organs. In the liver, E2 treatment was associated with lower acquisition of the capability to metabolize E2. In the ovary, E2 depleted the oocyte pool in a dose dependent manner by PND3. In 10 µg/day E2-treated ovaries, apoptotic oocytes were observed in newly formed follicles in addition to areas of ovarian cord remodeling. At PND6, follicles without any visible oocyte were present and multi-oocyte follicles were not observed. Our study reveals a major species-difference. Indeed, neonatal exposure to E2 depletes the oocyte pool in the rat ovary, whereas in the mouse it is well known to increase oocyte survival.
ISSN: 1932-6203

hal-00936582
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00936582
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00936582/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00936582/file/Systemic_Compensatory_Response-accepted.pdf
DOI : 10.1371/journal.pone.0082175

Éditeur(s) : HAL CCSD BioScientifica
Résumé : International audience
The general rules established from mammalian species for the regulation of spermatogenesis by gonadotropins may not be fully relevant in fish. Particularly, Fsh is as potent as Lh to stimulate steroidogenesis and the Fsh receptor is expressed in Leydig cells. In seasonal breeders, Fsh is likely the major gonadotropin involved in spermatogenesis onset and Lh is required to support spermatogenesis progression and gamete release. However, the genes that relay the action of Fsh and Lh have been poorly investigated in fish. The present study was aimed at identifying gonadotropin-dependent genes expressed in the testis during fish puberty. We cultured pubertal trout testicular explants for 96 h, with or without gonadotropin, and analyzed transcriptome variations using microarrays. Fsh and Lh had similar effects on a large group of genes while other genes were preferentially regulated by one or the other gonadotropin. We showed that most of the responsive genes were expressed in somatic cells and exhibited relevant patterns during the seasonal reproductive cycle. Some genes preferentially modulated by Lh could be involved in testicular cell fate (pvrl1 and bty) or sperm maturation (ehmt2 and racgap1) and will deserve further examination. Besides Fsh's effects on the steroidogenic pathway, our study demonstrates that Fsh coordinates relevant stimulatory and inhibitory paracrine factors known to regulate early germ cell proliferation and differentiation. Some of these genes belong to major regulatory pathways including the Igf pathway (igf1b/igf3 and igfbp6), the Tgfb pathway (amh, inha, inhba, and fstl3), the Wnt pathway (wisp1), and pleiotrophin (mdka).
ISSN: 0952-5041

inserm-00812401
http://www.hal.inserm.fr/inserm-00812401
DOI : 10.1530/JME-12-0197
PUBMED : 23045716

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Among pregnant women ibuprofen is one of the most frequently used pharmaceutical compounds with up to 28% reporting use. Regardless of this, it remains unknown whether ibuprofen could act as an endocrine disruptor as reported for fellow analgesics paracetamol and aspirin. To investigate this, we exposed human fetal testes (7-17 gestational weeks (GW)) to ibuprofen using ex vivo culture and xenograft systems. Ibuprofen suppressed testosterone and Leydig cell hormone INSL3 during culture of 8-9 GW fetal testes with concomitant reduction in expression of the steroidogenic enzymes CYP11A1, CYP17A1 and HSD17B3, and of INSL3. Testosterone was not suppressed in testes from fetuses younger than 8 GW, older than 10-12 GW, or in second trimester xenografted testes (14-17 GW). Ex vivo, ibuprofen also affected Sertoli cell by suppressing AMH production and mRNA expression of AMH, SOX9, DHH, and COL2A1. While PGE2 production was suppressed by ibuprofen, PGD2 production was not. Germ cell transcripts POU5F1, TFAP2C, LIN28A, ALPP and KIT were also reduced by ibuprofen. We conclude that, at concentrations relevant to human exposure and within a particular narrow â €' early window' of sensitivity within first trimester, ibuprofen causes direct endocrine disturbances in the human fetal testis and alteration of the germ cell biology. © The Author(s) 2017.
ISSN: 2045-2322

hal-01517095
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01517095
DOI : 10.1038/srep44184
PUBMED : 28281692

Éditeur(s) : Elsevier Science Inc
Résumé : The sex-determining system of Crassostrea gigas is still poorly known, especially regarding the potential influence of temperature. In order to address this question, mRNA expressions of actors of the molecular cascade (Cg-DMI, Cg-SoxE, Cg-beta-catenin, Cg-Foxl2/Cg-FoxI2os) and of Oyvlg, a germ cell marker, were investigated by real-time PCR in spat grown at different temperatures (18, 22, 25 and 28 degrees C). In parallel, gonadic differentiation, gametogenesis and sex ratios were assessed by histology at each of these temperatures. Whatever the temperature, Cg-DMI, Cg-SoxE, Cg-beta-catenin and Oyvlg expressions peaked at the same developmental stage, always after Cg-Foxl2/Cg-Foxl2os (around 40-44 dpf for spat grown at 18 degrees C). Temperatures increased the kinetics of first gonadic differentiation and gametogenesis. At 25 degrees C a significant switch occurred in sex ratio towards males and in the balance of expression between male and female genes, in favor of males. A slight gametogenesis disturbance was also observed. These results strengthen the hypotheses about the sex-determining time window and molecular cascade governing the development of C gigas, with notably the involvement of Cg-Foxl2/Cg-Foxl2os in the very early steps. They also suggest an influence of temperature on the oyster's sex determination which, associated to genetic control, would induce a mixed sex determination system (GSD +TSD). (C) 2013 Elsevier Inc. All rights reserved.
Comparative Biochemistry And Physiology A-molecular & Integrative Physiology (1095-6433) (Elsevier Science Inc), 2013-05 , Vol. 165 , N. 1 , P. 61-69

Droits : 2013 Elsevier Inc. All rights reserved
http://archimer.ifremer.fr/doc/00136/24767/22874.pdf
DOI:10.1016/j.cbpa.2013.02.007
http://archimer.ifremer.fr/doc/00136/24767/

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
About 1-6% of the genetic ancestry of modern humans today originates from admixture with archaic humans. It has recently been shown that autosomal genomic regions with a reduced proportion of Neanderthal and Denisovan ancestries (NA and DA) are significantly enriched in genes that are more expressed in testis than in other tissues. To determine whether a cellular segregation pattern would exist, we combined maps of archaic introgression with a cross-analysis of three transcriptomic datasets deciphering the transcriptional landscape of human gonadal cell types. We reveal that the regions deficient in both NA and DA contain a significant enrichment of genes transcribed in meiotic germ cells. The interbreeding of anatomically modern humans with archaic humans may have introduced archaic-derived alleles that contributed to genetic incompatibilities affecting meiosis that were subsequently purged by natural selection.
ISSN: 0737-4038

hal-01578587
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578587
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578587/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578587/file/J%C3%A9gou%20et%20al%20-%20Meiotic%20Genes%20Are%20Enriched.pdf
DOI : 10.1093/molbev/msx141

Éditeur(s) : HAL CCSD National Academy of Sciences
Résumé : International audience
Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the ``germ-line multipotency program'' and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells.
ISSN: 0027-8424

hal-01544480
https://hal.archives-ouvertes.fr/hal-01544480
DOI : 10.1073/pnas.1514789112

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
More than half the pregnant women in the Western world report taking mild analgesics. These pharmaceutical compounds have been associated with congenital cryptorchidism in humans, the best-known risk factor for low semen quality and testicular germ cell cancer. Furthermore, some of these mild analgesics exert potent anti-androgenic effects in the male rat and several endocrine-disrupting compounds, known to alter masculinization, have also been shown to be potent inhibitors of prostaglandin (PG) synthesis similar to mild analgesics. Using a 3-day ex vivo organotypic model system based on gestational day 14.5 rat testes, we herein show that testosterone production was inhibited by paracetamol, at doses of 0.1 μm to 100 μm. Similar results were obtained for aspirin (1-100 μm) and indomethacin (10 μm). The production of the other Leydig cell hormone, Insl3, was not disrupted by exposure to paracetamol. Investigations of the gross anatomy of the testis as well as Leydig cells number and rate of gonocyte apoptosis after the 3 days of ex vivo differentiation showed no significant effect of the analgesics tested compared with controls. These data indicate therefore that mild analgesics specifically inhibit testosterone production in rat foetal testes in vitro and that these compounds had no effect on gonocyte survival. Parallel determinations of prostaglandin D2 (PGD2) production indicated that the effects of paracetamol and aspirin on PGD2 and testosterone were not connected, whereas the effects of indomethacin were correlated. We conclude that mild analgesics exert direct and specific anti-androgenic effects in rat foetal testis in our experimental setup and that the mechanism of action is probably uncoupled from the inhibition of PG synthesis.
ISSN: 0105-6263

hal-00876956
https://hal.archives-ouvertes.fr/hal-00876956
DOI : 10.1111/j.1365-2605.2012.01282.x
PUBMED : 22612476

Éditeur(s) : HAL CCSD Elsevier Masson
Résumé : International audience
Parapneumonic pleural effusions represent the main cause of pleural infections. Their incidence is constantly increasing. Although by definition they are considered to be a “parapneumonic” phenomenon, the microbial epidemiology of these effusions differs from pneumonia with a higher prevalence of anaerobic bacteria. The first thoracentesis is the most important diagnostic stage because it allows for a distinction between complicated and non-complicated parapneumonic effusions. Only complicated parapneumonic effusions need to be drained. Therapeutic evacuation modalities include repeated therapeutic thoracentesis, chest tube drainage or thoracic surgery. The choice of the first-line evacuation treatment is still controversial and there are few prospective controlled studies. The effectiveness of fibrinolytic agents is not established except when they are combined with DNase. Antibiotics are mandatory; they should be initiated as quickly as possible and should be active against anaerobic bacteria except for in the context of pneumococcal infections. There are few data on the use of chest physiotherapy, which remains widely used. Mortality is still high and is influenced by underlying comorbidities.
Les épanchements pleuraux parapneumoniques représentent la principale étiologie des pleurésies infectieuses. Leur incidence est en constante augmentation. Bien qu’ils soient qualifiés de « parapneumoniques », l’épidémiologie microbienne est différente de celle des pneumonies avec notamment une plus grande fréquence des germes anaérobies. La ponction pleurale exploratrice représente l’étape diagnostique centrale qui permet de distinguer les épanchements parapneumoniques compliqués et non compliqués. Seuls les épanchements parapneumoniques compliqués doivent faire l’objet d’un traitement évacuateur reposant sur la mise en œuvre de ponctions pleurales répétées (PPR), d’un drainage thoracique ou d’une prise en charge chirurgicale. Le choix de la technique évacuatrice première reste débattu car il existe peu d’études prospectives comparatives. L’utilisation d’agents fibrinolytiques n’a pas fait la preuve de son efficacité sauf lorsqu’ils sont utilisés en association à la DNAse. L’antibiothérapie doit être systématique, précoce et couvrir les germes anaérobies, sauf en cas d’infection à pneumocoque. Il existe peu de données sur la place de la kinésithérapie qui reste largement utilisée. La mortalité est élevée et directement influencée par les comorbidités sous-jacentes.
ISSN: 0761-8425

hal-01117905
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01117905
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01117905/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01117905/file/Les%20%C3%A9panchements%20pleuraux%20parapneumoniques_accepted.pdf
DOI : 10.1016/j.rmr.2014.12.001

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
The strikingly complex structural organization of the mammalian testis in vivo creates particular difficulties for studies of its organization, function and regulation. These difficulties are particularly pronounced for investigations of the molecular communication networks within the seminiferous tubules that govern spermatogenesis. The use of classical molecular and cell biology approaches to unravel this complexity has proved problematic, due to difficulties in maintaining differentiated germ cells in vitro, in particular. The lack of a suitable testing ground has led to a greater reliance on high-quality proteomic and genomic analyses as a prelude to the in vitro antx1d in vivo testing of hypotheses. In this study, we highlight the options currently available for research, as used in our laboratory, in which proteomic and integrative genomic strategies are applied to the study of spermatogenesis in mammals. We will comment on results providing insight into the molecular mechanisms underlying normal and pathological spermatogenesis and new perspectives for the treatment of male infertility in humans. Finally, we will discuss the relevance of our strategies and the unexpected potential and perspectives they offer to teams involved in the study of male reproduction, within the framework of the Human Proteome Project. SIGNIFICANCE: Integrative genomics is becoming a powerful strategy for discovering the biological significance hidden in proteomic datasets. This work introduces some of the integrative genomic concepts and works used by our team to gain new insight into mammalian spermatogenesis, a remarkably sophisticated process. We demonstrate the relevance of these integrative approaches to understand the cellular cross talks established between the somatic Sertoli cells and the germ cell lineage, within the seminiferous epithelium. Our work also contributes to new knowledge on the pathophysiology of testicular function, with promising clinical applications. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.
ISSN: 1874-3919

hal-01063931
https://hal.archives-ouvertes.fr/hal-01063931
DOI : 10.1016/j.jprot.2014.04.013
PUBMED : 24751586

Éditeur(s) : HAL CCSD Society for the Study of Reproduction
Résumé : International audience
Mammalian spermatogenesis is a complex and highly orchestrated combination of processes in which male germline proliferation and differentiation result in the production of mature spermatozoa. If recent genome-wide studies have contributed to the in-depth analysis of the male germline protein-encoding transcriptome, little effort has yet been devoted to the systematic identification of novel unannotated transcribed regions expressed during mammalian spermatogenesis. We report high-resolution expression profiling of male germ cells in rat, using next-generation sequencing technology and highly enriched testicular cell populations. Among 20 424 high-confidence transcripts reconstructed, we defined a stringent set of 1419 long multi-exonic unannotated transcripts expressed in the testis (testis-expressed unannotated transcripts [TUTs]). TUTs were divided into 7 groups with different expression patterns. Most TUTs share many of the characteristics of vertebrate long noncoding RNAs (lncRNAs). We also markedly reinforced the finding that TUTs and known lncRNAs accumulate during the meiotic and postmeiotic stages of spermatogenesis in mammals and that X-linked meiotic TUTs do not escape the silencing effects of meiotic sex chromosome inactivation. Importantly, we discovered that TUTs and known lncRNAs with a peak expression during meiosis define a distinct class of noncoding transcripts that exhibit exons twice as long as those of other transcripts. Our study provides new insights in transcriptional profiling of the male germline and represents a high-quality resource for novel loci expressed during spermatogenesis that significantly contributes to rat genome annotation.
ISSN: 0006-3363

hal-01063924
https://hal.archives-ouvertes.fr/hal-01063924
DOI : 10.1095/biolreprod.114.118166
PUBMED : 24740603

Éditeur(s) : Gauthier-villars
Résumé : Until recently, the study of the pathology of oysters concerned some bacterial and a large number of parsitic diseases. Among the latter, diseases caused by fungi such as Labyrinthomyxa marina or by sporozoa such as Minchinia nelsoni in the American oyster have had very important econimic repercussions in the affected areas. The causes of some severe epizooties, such as the "Malpecue Bay disease" in Canada, have never been determined; in other cases, including the mass mortality of the Portuguese oyster in Fance, several years of investigations have been required before the first results of an etiologic nature were obtained. The use of electron microscopy has permitted great strides in the pathology of oysters. Thus, virus diseases have been found to be associated with the diseases of the Portuguse oyster, with gill disease and with the mass mortality of 1970. New and important data have also been obtained with regard to the structure and development of Marteilia refringens a parasite of the flat oyster which has been recognized as belonging to the haplosporidan species. Recently, Mycoplasma-like and Rickettsia-like organisms have been detected in several oyster species and oyster viruses have been isolated on cell cultures. Such results confirm the evolution of research in this field.
Les résultats acquis en matière de pathologie des huîtres concernaient jusqu'à une date récente quelques maladies bactériennes et surtout de nombreuses parasitoses. Pour certaines épizooties graves, comme la maladie de l'huître américaine de Malpèque au Canada, les causes n'ont jamais été déterminées et pour d'autres, comme la mortalité massive de l'huître portugaise en France, les premiers résultats d'ordre étiologique n'ont été obtenus que tardivement avec la mise en évidence d'une infection virale. L'étude ultra-structurale de Marteilia refringens, parasite de l'huître plate, la mise en évidence d'une virose associées à la maladie des branchies de l'huître portugaise, puis la découverte récente de nouveaux germes pathogènes tels que mycoplasmes et rickettsies et l'isolement de virus sur cultures cellulaires chez plusieurs espèces d'huîtres devaient confirmer l'évolution des recherches dans ce domaine.
Oceanologica Acta (0399-1784) (Gauthier-villars), 1978 , Vol. 1 , N. 2 , P. 255-262

Droits : 1978 Elsevier
http://archimer.ifremer.fr/doc/00015/12631/9520.pdf
http://archimer.ifremer.fr/doc/00015/12631/

Éditeur(s) : HAL CCSD Cold Spring Harbor Laboratory Press
Résumé : International audience
Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method--single-stranded DNA (ssDNA) sequencing (SSDS)--that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.
ISSN: 1088-9051

hal-00877675
https://hal.archives-ouvertes.fr/hal-00877675
DOI : 10.1101/gr.130583.111
PUBMED : 22367190