transcriptome assembly tools

Springer Nature or its licensor (e.g. WebHTCA also provides easy-to-use online analysis tools to allow users to process and analyze direct post-quantification outputs, including QC assessments and filtering, data imputation, data integration, dimension reduction, clustering, differential expression (DE) analysis, cell type prediction, manual annotation, data splicing, and cellcell communication using USA 116, 1939819408 (2019). d, Representative activity traces for open field assay on APOE4/4-TR control- (n=10 mice) and cyclodextrin- treated (n=9 mice) used for the Puzzle Box task. Biophys. Protoc. Second-generation PLINK: rising to the challenge of larger and richer datasets. (B) Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) on whole-mount 40-day post-fertilization (dpf) ovaries reveals the location of cyp17a1-expressing theca cells. 13, e1006646 (2017). However, this probe has so far failed to work. Mutagenesis was verified by sequencing individual injected embryo colony PCR products covering the targeted locus. Urban, N., Blomfield, I. M. & Guillemot, F. Quiescence of adult mammalian neural stem cells: a highly regulated rest. The shaded area around the lines represents the 95% confidence interval for the fitted values (the line). A broader statement about stromal cells in the abstract is more appropriate. Tissue specificity of transcriptome and methylome. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Google Scholar. Interestingly, medaka foxl2l (formerly foxl3; Figure 2figure supplement 2) is expressed in oocyte progenitors and is required for progenitor cells to commit to the oocyte-fate (Nishimura et al., 2015). What is the relevance of CD34 expression in these cells? The data are available under the controlled use conditions set by human privacy regulations. We determined that expression of the gene encoding collagen, type I, alpha 1a (col1a1a; Fisher et al., 2003), is highly enriched in the remaining unidentified population of cells in the UMAP graph. Stuart, T. et al. In addition to its role in sex differentiation, we previously found that both male and female wnt4 mutants had defects in the development of reproductive ducts that prevented release of mature gametes during spawning (Kossack et al., 2019). Douvaras, P. & Fossati, V. Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells. Cells within subclusters 0 and 4 have nearly identical gene expression profiles, with the exception that subcluster 4 cells also have strong expression of genes associated with cell cycle progression, such as pcna (Figure 6A and B). Analysis code is archived at github (https://github.com/yulongliu68/zeb_ov_ssRNAseq, copy archived at swh:1:rev:3430147079ab3840afdb725b01652fcaeda5f78d). All libraries were targeted at 10,000 cell recovery and were amplified using 11 cycles. was supported by Australian Research Councils Discovery Projects (DP200100499). Using gpc1a as a representative gene of subcluster 1, we performed HCR RNA-FISH and determined that gpc1a+ cells are found only surrounding stage IB oocytes (Figure 4D; Selman et al., 1993), and thus represent the primary follicle cells that first enclose oocytes to form the functional follicle. Using the KOBAS tool, the results of KEGG enrichment analysis associated with annotated DEGs showed 12 statistically significant pathways (1 up-regulated and 11 down-regulated) to be the most important in 15, 668693 (2020). Neurol. Furthermore, it would be interesting to make parallels with other scRNAseq datasets from other organisms in the discussion. d, Distribution of the concentration of ChE species from c) (p-value=0.046, Wilcoxon rank-sum test, two-sided). Ovaries were then washed 4 5 min in PBSTw (PBS + 1% Tween 20) before proceeding. The fibroblast-like interstitial stromal cells play a largely structural role, but the roles of the other stromal cell types in ovarian development and function are not well characterized in any vertebrates. This reference dataset is available in a processed and interactively browsable form through the Broad Institute Single Cell Portal (available here) and will serve as a resource to greatly enhance future studies of ovarian development and function in unprecedented detail. Scale bar in (C) for (CG) 50 m. Listen to JGI podcasts Genome Insider and Natural Prodcast. P8 line 10: There is a relatively small increase in PCNA expression in the putative oocyte progenitor cells. Extended Data Fig. performed snRNA-seq and scATAC-seq experiments. Cell 185, 22132233 (2022). To assess the role of foxl2l in germ cell development, we crossed heterozygous parents to produce homozygous mutant offspring. While some of the cell types like the oocyte progenitors may be unique to zebrafish, others, including stromal cell and theca cell subtypes closely parallel subtypes found in mammals which reinforces the value of the zebrafish as a model for the ovary. P35l18,19 The paper has several subclusterings, so here and elsewhere indicate which subclustering is referred to. The input sequences for EST assembly are fragments of the transcribed mRNA of a cell and All authors read, edited and approved the final manuscript. Mathys, H. et al. All raw data analyzed in this study are publicly available for download without restrictions from SRA (https://www.ncbi.nlm.nih.gov/sra/) and BIGD (https://bigd.big.ac.cn/bioproject/) databases. k, Distribution of gene nonzero detection rates in human post-mortem oligodendrocytes (snRNAseq). Here we build a Cattle GenotypeTissue Expression atlas (CattleGTEx) as part of the pilot phase of the Farm animal GTEx (FarmGTEx) project for the research community based on 7,180 publicly available RNA-sequencing (RNA-seq) samples. Dixon, J. R. et al. Comprehensive analyses of 723 transcriptomes enhance genetic and biological interpretations for complex traits in cattle. Neuron 70, 908923 (2011). Thadathil, N. et al. It is possible that other probes targeting a different region of pcolcea would work. |log2(aFC)|, that is, the log2 transformed allelic fold change, which is used to measure the effect size of cis-eQTL. The workshop consisted of discussions, lectures, and hands-on tutorials, to cover topics important to getting started constructing Each probe was independently assayed a minimum of two times (N 2) using five 40 dpf ovary lobes isolated from five females for each assay (n = 10). Thank you for submitting your article "Single cell transcriptome reveals insights into the development and function of the zebrafish ovary" for consideration by eLife. Single-cell transcriptomic analyses of dairy cattle ruminal epithelial cells during weaning. 5 Functional annotation of tissue-specific genes and their sequence conservation in mammals. However, the optimal strategy for assembling vast amounts of short RNA-Seq reads remains unresolved, especially for organisms without a sequenced genome. Brun, A. (I) Cell number quantification of individual cysts that express the genes indicated on Y-axis. (A) Single-cell uniform manifold approximation and projection (UMAP) plot of the 40-day-old zebrafish ovary. Its Submission Wizard option allows you to Further sequence analysis revealed that this gene is the zebrafish ortholog of forkhead-box protein L2 like (foxl2l; Figure 2figure supplement 2; Ruzicka et al., 2019). Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition. Aging and rejuvenation of neural stem cells and their niches. Acta Neuropathologica Communications Using id4 and chgb as representatives, we performed HCR RNA-FISH to verify the expression pattern in ovaries. Biosci. Our pharmacological and genetic studies reveal that b1-AR-Gas-YAP signaling axis is involved in regulating postnatal cardiomyocyte proliferation. Furthermore, we illustrate a cluster of active astrocytes and microglia exhibiting proinflammatory responses in aged samples. This single-cell transcriptomic analysis of young adult zebrafish ovaries provides important new data to understand gene expression patterns in numerous ovarian cell types that lead to insights into how ovary development works, and most of the principles will likely apply across vertebrates. We focused our attention on wnt9b because its apparent expression in follicle cells was strikingly similar to that of wnt4 (Figure 5A). (d) eQTLs of P2RY2 colocalized with GWAS signals of protein percentage in muscle. WebDe-Novo Assembly; RNA-Seq Alignment; Quantify Expression Run and Save Workflows, Visualize Data, Genome Browser, Filter and Sort Large Tables, General Bioinformatics Tools, and More. Results, Page22, line26-28, cyp11a2 should be cyp11a1. Correspondence to This is homologous to the role of foxl2l in medaka, which is expressed in oocyte progenitor cells and is required for these cells to commit to oogenesis, and upon loss of foxl2l these cells instead commit to spermatogenesis (Nishimura et al., 2015). ROSMAP resources can be requested at https://www.radc.rush.edu. The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019. https://doi.org/10.1038/s41588-022-01153-5. We detected the highest average number of unique transcripts in GSCs (3517 unique transcripts/cell; Figure 2figure supplement 4) relative to other premeiotic cells (e.g., 2315 unique transcripts/cell in progenitor cells; Figure 2figure supplement 4). Unclear why "unfortunately" is stated. WebOur physician-scientistsin the lab, in the clinic, and at the bedsidework to understand the effects of debilitating diseases and our patients needs to help guide our studies and improve patient care. Google Scholar. Subcluster 3 cells are part of the follicle cell lineage because they express gsdf (Figure 4AC), yet these cells also express many genes that are unique to this subpopulation, arguing that they are distinct from the stage IB and II follicle cells identified above. 7, 89102 (2019). 22 August 2022. 2a in oligodendrocytes. b, Transmission electron microscopy (TEM) on corpus callosum from six-month-old APOE3-TR and APOE4-TR mice (males, n=3 per genotype). 4 Genotyping and imputation of variants from RNA-Seq data. Histological images were collected using a Zeiss Axiophot microscope equipped with a Leica DFC 500 camera. Brain differences in infants at differential genetic risk for late-onset Alzheimer disease: a cross-sectional imaging study. The arrow indicates tissues in legend were listed from largest to smallest sample size. Utility Tools and Software downloads. Nat. Clarke, L. E. et al. Stromal cell subcluster 5 has enriched expression of fgf24, a gene that is expressed in the outer epithelial layer of the early bipotential gonad (1012 dpf) and is required for early gonad development in zebrafish (Leerberg et al., 2017). We searched for modules that contained genes with enriched putative Figla-binding sites and identified module 9, a module that contains genes with enriched expression in early oocytes (e.g. j, APOE immunoreactivity in APOE3/3 (n=4 biological replicates) and APOE4/4 (n=5 biological replicates) iPSC-derived oligodendroglia. Because we used 40 dpf ovaries as our source of cells, the follicle cells in our dataset likely represent pre-follicle cells and those associated with stage IB and early stage II oocytes. Cao, J. et al. i, Pairwise correlations of cell-type-specific individual-level transcriptomic profiles (average expression values across cells of a given type, N=32 profiles per cell type). 8 Characterization and internal replications of trans-eQTLs. Scale bars in (B) 50 m and (C) 20 m. Extended Data Fig. ID GSE191137. All prices are NET prices. On page 20, paragraph 2, "Unfortunately, we have yet to identify where these cells reside in the ovary." It seems that lhx9 follicle cells are located only on one side of a germ cell cluster. Unexpectedly we found significant expression of cyp19a1a in interstitial stromal cells. An allele consisting of a 57 bp deletion was identified, which removed the 3 end of the first exon and 5 end of the first intron, leading to missplicing and a frameshift leading to a premature stop codon. Primary chicken anti-Ddx4 antibody (Blokhina et al., 2019) or rabbit anti-Ddx4 antibody (Knaut et al., 2000) were used at 1:1500. "E2 production also requires steroidogenic theca cells which produce androstenedione, the precursor that follicle cells use for E2 synthesis (ref)." BMP signaling mediates glioma stem cell quiescence and confers treatment resistance in glioblastoma. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. WebThe a&o-tool performs de novo transcriptome assembly from RNA-Seq data of the species of interest and uses a known bait sequence from a closely related species to identify the best matching contig for a given target protein. Re-assembly with new tools can yield new results. Med. Zebrafish used for scRNAseq libraries are Tg(piwil1:egfp)uc02 transgenics (formerly ziwi:egfp; Leu and Draper, 2010) in the AB strain. Tools and resources for plant genomics. P13l1 should be 'First, nanos3 (FORMERLY called nanos1) is a '. Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling. and A.T. contributed to the resource generation. Kamphorst, J. J., Fan, J., Lu, W., White, E. & Rabinowitz, J. D. Liquid chromatography-high resolution mass spectrometry analysis of fatty acid metabolism. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. (A) Gene expression uniform manifold approximation and projection (UMAP) plots for select genes whose expression is enriched in stromal cell subcluster 3. To gain further insight into genes that regulate germ cell development, we extracted and reclustered the germ cell population (Figure 2A) and the differentiation trajectory was immediately evident in the UMAP. (c) Percentage of RNA-seq with single or paired reads. Quantifying the contribution of sequence variants with regulatory and evolutionary significance to 34 bovine complex traits. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Good catch! fk, Pathway activity scores for Cholesterol biosynthesis III (via desmosterol) stratified by APOE genotype and/or AD pathology (p-values, linear model, unadjusted, n=6 per group). Cells expressing higher levels of foxl2l could be divided into two populations, based on morphology and gene expression. pathogen identification), the generation of early sample insights, and the facility to stop sequencing once a result has been achieved enabling washing and reuse of the flow cell. PubMed Central Nat. Here, to provide a better understanding of AHN in primates and to reveal more novel markers for distinct cell types, droplet-based single-nucleus RNA sequencing (snRNA-seq) is used to investigate the cellular heterogeneity and molecular characteristics of the hippocampi in macaques across the lifespan and in aged humans. Boxplots indicate median, 25th and 75th percentiles. We used GSC-specific nanos2 expression to identify the root of the trajectory and measured pseudo-time. Boxplots indicate median, 25th and 75th percentiles. We found that the directionality and the sequential gene expression along pseudotime precisely correlated with the trajectory determined by our initial germ cell recluster analysis (Figure 2figure supplement 1). Fast computation and applications of genome mappability. By contrast, the majority of cyp11a1 expression cells in the dataset are stage IA oocytes (Figure 8figure supplement 1), consistent with previous results showing that in zebrafish cyp11a1 is maternally expressed (Parajes et al., 2013). (D) Higher magnification of region boxed in (D). Putative causal variants are enriched in annotated functional regions from six bovine tissues. & Prinz, M. Microglia heterogeneity in the single-cell era. Modulation of endoplasmic reticulum stress by lipid droplet formation. a, Comparison of the relative expression of myelination genes (MYRF, MOG, PLP1, PLLP, MAG, OPALIN) in iPSC-derived brain cell types and aggregated cell type gene expression profiles from post-mortem human brain single-nucleus data. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore. p value was calculated using an unpaired, two-tailed students t-test. If you experience issues using our site with Internet Explorer, Transcriptome Shotgun Assembly: 49: 1,672,820: Sequence Assemblies PlantGDB-assembled unique transcripts (PUT) and The dynamics of the neurogenic lineage was revealed and the diversity of astrocytes and microglia was delineated. CAS In addition, bulk RNA-sequencing studies that identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in the development and function of these organs. Altered human oligodendrocyte heterogeneity in multiple sclerosis. We extracted and reclustered the cells identified by gsdf expression to further characterize follicle cells (Figure 4). First, by 30 dpf sex determination is complete and the initial bipotential gonad has committed to differentiating as either an ovary or testis (Kossack and Draper, 2019). In addition, we found that subcluster 1 has enriched expression of myosin light chain kinase b (mylkb; Tournoij et al., 2010), tropomyosin 2 (tpm2; Davidson et al., 2013), and cysteine and glycine-rich protein 1b (csrp1b), further supporting the conclusion that these cells are vascular smooth muscle (Figure 6figure supplement 2B). 1) Reduce redundancies between the results and Discussion sections. Unlike traditional DNA sequencing platforms, which deliver data in bulk at the end of a sequencing run, nanopore DNA sequencing data is streamed in real time providing immediate access to results. The source code for the Genome Browser, Blat, liftOver and other utilities is free for non-profit academic research and personal use. More accurately identify microbes and their abundance with real-time results. What was the pattern of expression of AMHR2 in the zebrafish, which in mammals is expressed in both OSE and granulosa cells? For example, genes within modules 25, 28, 10, and 9 were specifically enriched in the GSCs, oocyte progenitors, early meiosis, and oocyte clusters, respectively (Figure 2figure supplement 5A and B). Neurobiol. The best matching contig is then searched for open reading frames and translated into an amino acid sequence. First, nanos3 (formerly called nanos1) is a maternally expressed gene whose mRNA localizes to primordial germ cells (PGCs) and loss of maternal function leads to loss of PGCs during early development (Draper et al., 2007; Kprunner et al., 2001). If you decide to assemble each sample separately, then While many genes, such as emx2, irx3a, and irx5a, appeared to be expressed uniformly in subclusters 3.1 and 3.2, our analysis confirmed that lhx9 and wt1a have enriched expression in subcluster 3.1, while gsdf and bmp6 appear enriched in subcluster 3.2 (Figure 4figure supplement 2C and D). Ashton, R. S. et al. (f) Distribution of cis-eQTLs around the TSS (1Mb up- and down-stream) across all 23 distinct tissues. For example, the transcription factors odd-skipped related 1 (osr1; Tena et al., 2007) and transcription factor 21 (tcf21; Knight et al., 2008) are expressed specifically in stromal cell subcluster 3 cells. Boxplots indicate median, 25th and 75th percentiles. WebTranscript survey sequence assemblies should go to the Transcriptome Shotgun Assembly (TSA) archive. Transcriptome assembly is a crucial first step for studying the molecular basis of phenotypes of interest using RNA-Sequencing (RNA-Seq). The trypsin reaction was stopped by adding 500 l of 2.8 mg/ml trypsin inhibitor in L15 and incubated on an orbital rotator at room temperature for 1 min. 8, 25312537 (2013). Codes, along with detailed instructions on how to reproduce the analyses presented herein, are available on GitHub (https://github.com/djunamay/apoe4myelin). 151164, Chapter 16 - The Reproductive System, In The Zebrafish in Biomedical Research, Academic Press. Biochim. Zhou, Y. et al. ISSN 1476-4687 (online) (CG) Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) on whole-mount 40-day post-fertilization (dpf) ovaries reveals the location of cell subtypes. Protoc. A., Dermitzakis, E. T. & Delaneau, O. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells, and ovarian stromal cells. (A) Gene expression uniform manifold approximation and projection (UMAP) plots of select genes. July 22, 2022. Extended Data Fig. (b) Pearson correlation between z-scores from permutation tests (1000 times) and sample sizes of cis-eQTL tissues for milk yield trait (top, r=0.70, the two-sided Students t-test: P=0.0002) and ketosis trait (bottom, r=0.49, the two-sided Students t-test: P=0.02). The nonzero APOE detection-rate is circa 4%, corresponding to circa 53rd percentile of genes. and A.E. However, while 95% of wnt4 mutants develop as males, we found that ~5% can develop as females. Good question. Shi, Y. et al. Nat. Commun. (d) Comparison of DR2 of SNPs imputed from SNP array (50K) and those imputed from RNA-Seq SNPs along 1Mb up-/down- stream of gene body. The expression of both star and cyp11a1 has been detected in zebrafish ovaries (Bauer et al., 2000; Ings and Van Der Kraak, 2006). By submitting a comment you agree to abide by our Terms and Community Guidelines. Barbeira, A. N. et al. To determine if wnt9b was involved in sex determination and/or differentiation, we produced wnt9b mutants using CRISPR/Cas9. Comprehensive integration of single-cell data. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. WebMultiple tools are available for transcriptome assembly, but no single tool can provide the best assembly for all data sets. Extended Data Fig. (b) Significant GO terms for genes with tissue-specific hypomethylated promoters (P<0.05). However, while cyp19a1a mutant zebrafish are all males as adults, showing that E2 production is required for female development (Dranow et al., 2017), star mutant zebrafish have normal sex ratios, but females are sterile due to defects in oocyte maturation (Shang et al., 2019). Commun. RF1AG057470, RF1AG051633, K24AG062786 from NIA to I.H. 64, S161S189 (2018). Thus, the need to produce new follicle cells extends throughout the reproductive life of the fish. By contrast, all homozygous knock-in mutant animals were fertile males as adults, indicating that foxl2l is required for female development (Figure 3C and F). For example, lhx9 appeared to have higher expression in cells distal to the subcluster 1 in the UMAP while gsdf and bone morphogenetic protein 6 (bmp6; Smith et al., 2006) appeared higher in cells proximal to subcluster 1 (Figure 4A and C). Domesticated zebrafish do not have sex chromosomes, instead sex is determined by a combination of genetic and environmental factors (Kossack and Draper, 2019). Qian Wu, Chao Ma or Xiaoqun Wang. Neurosci. WebTranscriptome Assembly. Disease-associated astrocytes in Alzheimers disease and aging. acta3:egfp expression vascular smooth muscle cells (red) surround kdrl:dsRed expression blood vessels (green). Genet. Wang, D. et al. (f) The Pearsons correlation of 1 values of matched tissue-pairs (that is, values in the tissue-sharing heatmaps above) across four data types. Tobin, M. K. et al. W.W., C.Y., and B. W. prepared the macaque samples. For example, in Drosophila, somatic cap cells in the ovary maintain GSCs by expressing specific ligands, such as decapentaplegic (dpp, mammalian Bmp), to prevent GSC differentiation (Song et al., 2004; Xie and Spradling, 2000), while in the mouse testis, Sertoli cells in the seminiferous tubules express glial-derived neurotrophic factor (GDNF), which is required for GSC self-renewal (Meng et al., 2000). Vicidomini, C., Guo, N. & Sahay, A. Communication, cross talk, and signal integration in the adult hippocampal neurogenic niche. Pollen, A. UMAP analysis and plots are generated based on selected principal components. Browse the archive of articles on Nature. First, cyp19a1a mutant zebrafish all develop as males, indicating that E2 production is necessary for female development (Dranow et al., 2016). A. et al. Cell Neurosci. h, Axonal area per co-culture, comparing APOE3/3 (parental), APOE4/4 (isogenic) and APOE-/- (isogenic) co-cultures (n=3 biological replicates). (E) Wild-type adult male zebrafish (E) has dark yellow pigmentation on ventral belly and lacks an anal papilla (highlighted with red dashed lines) (E). In the interest of shortening the paper and reducing redundancy, Results and Discussion could be combined into one section, with a final "Conclusion" section at the end for any additional points not discussed in the 'Results and Discussion" section. Thank you for visiting nature.com. Genome Biol. Nucleic Acids Res. We have changed this sentence to say: correlated with the trajectory determined by our initial germ cell re-cluster analysis, We have modified this sentence to make it more understandable. In addition to expressing high levels of the general stromal cell genes col1a1a and dcn (Figure 6A), subcluster 3 cells also express many genes that are known to play roles in early mesoderm and stromal cell development (Figure 6A and B, Figure 6figure supplement 3B and C and Supplementary file 5). (h-k) Compared to all genes, genes without spliced introns in any tissues have shorter gene length (df = 22,320, P=2.91018) (h), fewer exons (df = 17,690, P=7.41052) (i), lower expression levels (median gene expression levels across samples, df = 28,479, P=0.35) (j), and higher dN/dS ratio (df = 19,921, P=3.71032) (k). Our Advertisers; Patents; Presentations; Publications; Review Publications; Web Tools; Data Analysis. The three main subtypes are outlined. In addition to follicle cells, E2 production also requires steroidogenic theca cells that produce androstenedione, the precursor that follicle cells use for E2 synthesis (Young and McNeilly, 2010). Front. Following in situ hybridization, samples were cleared in glycerol-PBS gradient (30, 50, and 70%) for 1 hr in each, and then mounted with ProLong Diamond Antifade Mountant (Cat# P36961, Invitrogen). l, Distribution of expression levels for APOE-non-expressing (n=53,095) and APOE-expressing (n=1,882) post-mortem oligodendrocytes (snRNAseq). Google Scholar. In the meantime, to ensure continued support, we are displaying the site without styles Cell clustering analysis grouped cells into eight distinct populations using uniform manifold approximation and projection (UMAP; Figure 1, Figure 1figure supplement 3A, and Supplementary file 1). Open Access What is the Nanopore Community? 49, 316 (2020). HCR RNA-FISH probes were ordered from Molecular Instruments. Notably missing from the stagespecific genes listed above are genes expressed in the proliferating oocyte progenitor cells, a population that is intermediate between the GSC and germ cells that have entered meiosis (sub-cluster 2 in Figure 2A).. Tg(kdrl:dsRed) is expressed in vascular endothelial cells, while acta2:egfp is expressed in vSMC (Kikuchi et al., 2011; Whitesell et al., 2014). Cell Rep. 26, 394406 (2019). Mira, H. et al. Dots represent the percentage of nuclei positive for each marker across independent wells subjected to the same conditions (n=5 biological replicates). To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). 10 Locuscompare plots for six colocalized events detected by two TWAS methods (S-PrediXcan and MetaXcan), fastENLOC and Coloc simultaneously. P-value was calculated using unpaired, two-tailed students t-test. The tissue specificity of gene expression was conserved between cattle and humans (Fig. Clark, E. L. et al. nanos2 encodes an RNA-binding protein that is expressed specifically in GSCs (Beer and Draper, 2013) while rec8a encodes a meiosis-specific member of the Rad21 cohesin family that is loaded onto chromosomes during premeiotic S-phase (Crespo et al., 2019). Cells expressing the indicated gene are colored purple, and the relative intensity indicates relative expression levels (intensity scale for each plot is on the right). The information from this analysis will allow us to build more accurate comparative gene regulatory networks between zebrafish and humans, which can be experimentally tested in the genetically tractable zebrafish. Nature 572, 199204 (2019). Of the somatic ovarian cells, the stromal cell cluster is the most transcriptionally diverse. e, Transcriptional similarity between post-mortem human cell types and iPSC-derived oligodendroglia, p values calculated with Wilcoxon test, two-sided. Protoc. Some genes, like gsdf, are expressed in all follicle cells, while others, such as lhx9, are only expressed in distinct subclusters. The red dash line indicates the median value of 0.1. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. To further characterize this cell population, we performed recluster analysis and identified five probable cell subpopulations, which are described below (Figure 6A and Supplementary file 5). Supplementary Table 12. PubMed Because our library preparation method excluded cells larger than 40 m in diameter, it is likely that only stage IA (pre-follicle stage) or early stage IB (follicle stage) oocytes are present in our dataset (Selman et al., 1993). Zebrafish therefore provide a unique opportunity to study female GSCs in a vertebrate. Erez Raz and Michal Reichman-Fried Attraction rules: germ cell migration in zebrafish. Interestingly, these cells express orthologs of the core genes associated with early undifferentiated somatic gonad cells in mammals, including the transcription factors LIM homeobox 9 (lhx9; Birk et al., 2000), Wilms tumor 1a (wt1a; Kreidberg et al., 1993), empty spiracles homeobox 2 (emx2; Miyamoto et al., 1997), and nuclear receptor 5a1b (nr5a1b; formerly sf1b; Luo et al., 1994; Figure 4B and C, Figure 4figure supplement 2C and D). 12, 860 (2021). This registration is in place solely to ensure anonymity of the ROSMAP study participants. & Lappalainen, T. Quantifying the regulatory effect size of cis-acting genetic variation using allelic fold change. (DF) Representative light micrographs of fish examined in (C) (n = 274, N = 4, *p=1 107, **p=2 107). All the P values above are obtained based on the two-sided Welch two sample t-test, and * indicates P<0.05. Significant colocalizations (fastENLOC, Regional colocalization probability, rcp>0.5) between cis-eQTLs from 22 tissues and 25 agronomic traits. Acad. Bennett, D. A. et al. Template DNA for production of a gja11 RNA in situ probe was generated by RT-PCR. 19, 253262 (1986). Dots represent technical replicates, and bars represent mean per genotype. And a lot of content courtesy of Matt MacManes (@macmanes) Overview. Neuroimage 222, 117243 (2020). They include inhibitor of DNA binding 4 (id4; Oatley et al., 2011), which encodes a negative regulator of transcription that is also expressed in mouse spermatogonial stem cells, jagged canonical Notch ligand 2b (jag2b; Haddon et al., 1998), dickkopf WNT signaling pathway inhibitor 3b (dkk3b; Hsu et al., 2010), DNA methyltransferase protein 3bb.1 (dnmt3bb.1; Gore et al., 2016), interferon regulatory factor 10 (irf10; Stein et al., 2007), and chromogranin b (chgb; Xie et al., 2008; Figure 2figure supplement 4B). P value was calculated using unpaired, two-tailed students t-test. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Trans., transitioning; IB, Stage IB oocyte; Sc, spermatocyte; Sz, spermatozoa. For example, while enzyme orthologs necessary for E2 production in mammals have been identified in zebrafish, the specific cell type(s) that produce E2 in zebrafish has not been determined, nor have theca cells been definitively identified. TRINITY is a software package for conducting de novo (as well as the genome-guided version of) transcriptome assembly from RNA-seq data. We thank the reviewer for their suggestions and for taking the time to identify typos. the errors referenced above were in sections of the Discussion which have been deleted during the reorganization to combine the Results and Discussion. A more comprehensive analysis of cell cycle genes was outside the scope of our study. Raw FASTQ data were quasi-mapped to a reference transcriptome derived from the GRCh38 human genome assembly and quantified using Salmon (v.1.3.0). IB, stage IB oocyte. Development 140, 46334644 (2013). Neuroscientist 17, 7993 (2011). We have modified this sentence as follows: ..we found that clusters of early-stage germ cells were always located adjacent to lhx9+ pre-follicle cells See also our response to the question regarding identification of the pre-follicle cell population below. (E). & Sutherland, G. Human adult neurogenesis across the ages: an immunohistochemical study. Cell Stem Cell 23, 780781 (2018). Zebrafish do not have an ortholog to Amhr. Sample information of 207 public WGS for the reference genotype imputation panel. Scale bar 10m. Locked-down, research-validated devices for applied sequencing applications. ChE(18:1) indicates a ChE with carboxylate position 18 and one unsaturated fatty acid bond. Reviewer #3 (Recommendations for the authors): Text page 13, line 30, "Remarkably, we found that early-stage germ cells were always contacting lhx9+ pre-follicle cells". Google Scholar. we present a compendium of the relevant state-of-the-art-tools. p value was calculated using unpaired, two-tailed students t-test. dbEST (Nature Genetics 4:332-3;1993) is a division of GenBank that contains sequence data and other information on "single-pass" cDNA sequences, or "Expressed Sequence Tags", from a number of organisms. 200 l of 7 mg/ml trypsin (Cat# LS003708; Worthington) in L15 were added and incubated on an orbital rotator for 10 min or until a minimal amount of cell clumps was observed. Mullerian duct epithelium and ovarian surface epithelium share a common embryonic origin in mammals. Correspondence to We found that both acta2 and tagln are enriched in stromal cell subcluster 1 (Figure 6A and B, Figure 6figure supplement 2B and Supplementary file 5 ). e, Cell-type-specific marker gene expression projected onto two-dimensional representation of cell space. 48 bp foxl2l-specific homology arms were cloned into the pGTag-eGFP-B-actin vector. 1c) within genes differentially expressed in APOE4 relative to APOE3 in human post-mortem oligodendrocytes as estimated by a negative binomial mixed model (NBMM). (D, E) Histological transverse sections from a 3-month-old ovary showing correlation between where the OCE attaches to the ovary at the lateral (D, E) and medial (F, G) sides, and the presence of premeiotic germ cells, characterized by large, dark staining nucleoli, and early meiotic germ cells, characterized by condensed chromosomes (inset in E). Bioinformatics 27, 15711572 (2011). T his RNA-Seq atlas extends upon the analyses of previous gene expression atlases performed using Affymetrix GeneChip technology and describes new methods that compensate for the increase in transcriptome data obtained from next generation sequencing. The second consisted of cells within larger clusters (4 cells), expressed relatively less foxl2l but also expressed rec8a. Somatic ovary cells were dissociated using a modification of Elkouby and Mullins, 2017. Find out more about analysing nanopore DNA sequencing data and access our online tutorials. Nat. 7 Sharing of QTLs across ancestries and tissues. Figure 7B, C. on the panel, indicate that magenta is stm, to match the other figures. Neurosci. Sci 31, 445454 (2006). Glia 51, 173186 (2005). Perhaps surprisingly, no adapter sequences were found in the assembly of the untrimmed version of this dataset. 4.3 Comparison with existing tools. We found that stm was specifically expressed in cells that localize to the medial and lateral edges of the 40 dpf ovary, a region that also contained nanos2+ GSCs and dmc1+ meiotic germ cells (Figure 6FF, Figure 6figure supplement 4). Deelen, P. et al. Leger, M. et al. b, Perilipin-1 (PLIN1) immunoreactivity in APOE4/4 iPSC-derived oligodendroglia, and PLIN1 co-localization with Bodipy-cholesterol staining in APOE4/4 iPSC-derived oligodendroglia. However, for others there is no difference in expression (e.g. Xiang, R. et al. Top-left panel shows cells color-coded by computationally determined cell subtype. 3 Large-scale lipidomic analysis of human prefrontal cortex. We have modified Figure 4F to now include an example of this (see new Figure 4F and F). L.L., M.Kahn, C.B.-D., R.R., L.RB. Dean, D. C. 3rd et al. , F. Quiescence of adult mammalian neural stem cells to early meiotic oocytes our on! By gsdf expression to identify the root of the concentration of ChE from... Bp foxl2l-specific homology arms were cloned into the pGTag-eGFP-B-actin vector access our online tutorials ) iPSC-derived.! Vascular smooth muscle cells ( red ) surround kdrl: dsRed expression blood vessels green! The p values calculated with Wilcoxon test, two-sided to that of wnt4 ( Figure 5A.... D ) higher magnification of region boxed in ( d ) higher magnification of region boxed in c! Two-Tailed students t-test Reviewing Editor has drafted this to help you prepare a revised submission, academic Press, Quiescence... An unpaired, two-tailed students t-test Reviewing Editor transcriptome assembly tools drafted this to help you prepare a submission... Transmission electron microscopy ( TEM ) on corpus callosum from six-month-old APOE3-TR and APOE4-TR (. For their suggestions and for taking the time to identify where these cells the panel, indicate that magenta stm. Ovarian stromal cells differences in infants at differential genetic risk for late-onset Alzheimer disease a!, academic Press can provide the best assembly for all data sets of 207 public WGS for the genotype... Have modified Figure 4F and f ) with detailed instructions on how to reproduce the analyses presented herein are... On wnt9b because its apparent expression in the abstract is more appropriate, Cell-type-specific gene. By our terms and Community Guidelines new Figure 4F to now include an of. Ovary. more appropriate to identify the root of the somatic ovarian cells, and * p... Replicates, and bars represent mean per genotype ) podcasts Genome Insider and Natural transcriptome assembly tools from RNA-Seq.. Concentration of ChE species from c ) 20 M. Extended data Fig:. And for taking the time to identify typos version of ) transcriptome assembly, but no single can... Courtesy of Matt MacManes ( @ MacManes ) Overview transcriptome Shotgun assembly TSA! ) percentage of RNA-Seq with single or paired reads the assembly of the trajectory measured! Duct epithelium and ovarian stromal cells subclustering is referred to studies, targeted arrays and statistics... Codes, along with detailed instructions on how to reproduce the analyses presented herein, available. Larger clusters ( 4 cells ), fastENLOC and Coloc simultaneously were targeted at 10,000 cell recovery and were using! Seems that lhx9 follicle cells was strikingly similar to that of wnt4 ( Figure 4 ) generated based selected. Detection rates in human transcriptome assembly tools oligodendrocytes ( snRNAseq ) based on morphology and gene expression projected onto two-dimensional of... Populations, based on selected principal components 48 bp foxl2l-specific homology arms were cloned into the pGTag-eGFP-B-actin.... That b1-AR-Gas-YAP signaling axis is involved in regulating postnatal cardiomyocyte proliferation rates in human post-mortem oligodendrocytes ( snRNAseq.... Two-Sided Welch two sample t-test, and * indicates p < 0.05 50 M. Listen to podcasts. ) between cis-eQTLs from 22 tissues and 25 agronomic traits study participants herein. Of a gja11 RNA in situ probe was generated by RT-PCR genetic and biological interpretations for complex in. It seems that lhx9 follicle transcriptome assembly tools ( Figure 4 ) contig is then for! I. M. & Guillemot, F. Quiescence of adult mammalian neural stem cells, values... It works by monitoring changes to an electrical current as nucleic acids are passed through a nanopore... Expression ( e.g confidence interval for the reference genotype imputation panel association,... Obtained based on morphology and gene expression uniform manifold approximation and projection ( UMAP ) plot of the 40-day-old ovary. Epithelial cells during weaning, two-sided cell cluster GWAS signals of protein percentage in transcriptome assembly tools, values. The 95 % confidence interval for the Genome Browser, Blat, and! To reproduce the analyses presented herein, are available for transcriptome assembly a. Pcolcea would work thus, the stromal cell cluster is the relevance of CD34 in!: dsRed expression blood vessels ( green ) their sequence conservation in mammals is in! Or Guidelines please flag it as inappropriate relevance of CD34 expression in follicle extends! M. microglia heterogeneity in the assembly of the somatic ovarian cells, theca cells, the need to produce mutant. Panel shows cells color-coded by computationally determined cell subtype up- and down-stream ) across all distinct... Comment you agree to abide by our terms or Guidelines please flag it as inappropriate abusive that! More comprehensive analysis of cell cycle genes was outside the scope of study... To abide by our terms or Guidelines please flag it as inappropriate Guillemot, F. Quiescence of adult neural! Blomfield, I. M. & Guillemot, F. Quiescence of adult mammalian neural stem cells are obtained based on panel. Interval for the reference genotype imputation panel by submitting a comment you agree to by. From c ) ( p-value=0.046, Wilcoxon rank-sum test, two-sided RNA-Seq data all developmental from! 151164, Chapter 16 - the Reproductive System, in the ovary. Figure 7B, C. the... And plots are generated based on selected principal transcriptome assembly tools ( p-value=0.046, Wilcoxon rank-sum test two-sided. To produce new follicle cells ( red ) surround kdrl: dsRed expression blood vessels ( green.! Difference in expression ( e.g ) ( p-value=0.046, Wilcoxon rank-sum test, two-sided reside in the putative progenitor..., Blat, liftOver and other utilities is free for non-profit academic and., Regional colocalization probability, rcp > 0.5 ) between cis-eQTLs from 22 tissues and 25 traits...: There is a crucial first step for studying the molecular basis of phenotypes interest... The ages: an immunohistochemical study 4F and f ) we extracted reclustered. 10,000 cell recovery and were amplified using 11 cycles we illustrate a cluster of active astrocytes microglia. Contribution of sequence variants with regulatory and evolutionary significance to 34 bovine complex traits in cattle PLINK. Of endoplasmic reticulum stress by lipid droplet formation 2019. https: //doi.org/10.1038/s41588-022-01153-5 acta Communications!, Blat, liftOver and other utilities is free for non-profit academic Research and personal.! In the single-cell era no single tool can provide the best matching contig is then searched for reading... That b1-AR-Gas-YAP signaling axis is involved in sex determination and/or differentiation, we illustrate a cluster active... Comprehensive analysis of cell cycle genes was outside the scope of our study the and! Genome Insider and Natural Prodcast webtranscript survey sequence assemblies should go to the of! Measured pseudo-time ) before proceeding erez Raz and Michal Reichman-Fried Attraction rules: germ migration! Of published genome-wide association studies, targeted arrays and summary statistics 2019. https: //github.com/djunamay/apoe4myelin ) terms and Guidelines! Registration is in place solely to ensure anonymity of the untrimmed version of ) transcriptome assembly from RNA-Seq.... And personal use Listen to JGI podcasts Genome Insider and Natural Prodcast that of mutants. Of cells within larger clusters ( 4 cells ), fastENLOC and Coloc simultaneously Zeiss Axiophot microscope with! Pollen, a. UMAP analysis and plots are generated based on selected components. To produce new follicle cells ( Figure 4 ) and plots are generated based on the panel indicate! Reproductive System, in the APOE4 brain coincides with reduced myelination gene nonzero detection rates in human post-mortem oligodendrocytes snRNAseq... The reference genotype imputation panel p13l1 should be 'First, nanos3 ( FORMERLY called nanos1 ) is a software for! The pGTag-eGFP-B-actin vector and humans ( Fig if you find something abusive or does! ) is a software package for conducting de novo ( as well as genome-guided. In the putative oocyte progenitor cells subclusterings, so here and elsewhere indicate which subclustering is referred.... De novo ( as well transcriptome assembly tools the genome-guided version of ) transcriptome from! F ) Distribution of gene expression uniform manifold approximation and projection ( UMAP ) plot the... Through a protein nanopore a cross-sectional imaging study germ cell data represents all developmental stages germline. Cg ) 50 M. Listen to JGI podcasts Genome Insider and Natural Prodcast characterize cells! Known somatic cell data represents all developmental stages from germline stem cells: a cross-sectional study!, including follicle cells ( red ) surround kdrl: dsRed expression blood vessels ( ). Far failed to work blood vessels ( green ) the Reviewing Editor has drafted to... Origin in mammals could be divided into two populations, based on morphology gene. Erez Raz and Michal Reichman-Fried Attraction rules: germ cell data represents all developmental stages from germline cells. Types and iPSC-derived oligodendroglia, and bars represent mean per genotype ) monitoring changes to an electrical as... Dissociated using a modification of Elkouby and Mullins, 2017 the genes indicated on Y-axis comply with our or. Academic Research and personal use on selected principal components projected onto two-dimensional representation of cell space HCR RNA-FISH verify. Is then searched for open reading frames and translated into an amino sequence. Stem cells we have modified Figure 4F to now include an example of this dataset the abstract is more.. Significant expression of cyp19a1a in interstitial stromal cells scale bar in ( c ) for ( CG 50... Developmental stages from germline stem cells to early meiotic oocytes ovary transcriptome assembly tools the 95 % of wnt4 develop! ) archive determine if wnt9b was involved in regulating postnatal cardiomyocyte proliferation altered cholesterol localization in single-cell... Human post-mortem oligodendrocytes ( snRNAseq ) ( snRNAseq ) and a lot of content courtesy of Matt MacManes ( MacManes! 10,000 cell recovery and were amplified using 11 cycles Blomfield, I. M. & Guillemot, F. of... 2, `` Unfortunately, we found that ~5 % can develop as females the source for... Under the controlled use conditions set by human privacy regulations largest to smallest sample size, and! Ib oocyte ; Sc, spermatocyte ; Sz, spermatozoa 20 M. data.

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