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Brinkman EK, Chen T, Amendola M, Steensel B. & Bader, G. D. Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. Cells were passaged by diluting cultures 1:6 every day. Given the potential importance of RAD51B in processing damaged DNA in haploid leukemia, we asked if RAD51B and the HR pathway were involved in the in vivo response to DNA-damaging chemotherapy in near-haploid B-ALL. Khatri, P., Sirota, M. & Butte, A. J. Nat. Computational correction of copy number effect improves specificity of CRISPRCas9 essentiality screens in cancer cells. Protoc. 2018;5:68. Nucleic Acids Res. *: Students t-test p-value=0.026 (n=3), ***: Students t-test p-value=0.0002 (n=3). Kandoth, C. et al. More powerful procedures for multiple significance testing. Interestingly, despite elevated RAD51 expression in response to therapy in near-haploid but not diploidized B-ALL cells, both RAD51B expression and its associated expression signature score are relatively low in near-haploid vs. diploidized cells across all conditions (Supplementary Figure 3b). Bioinformatics 26, 139140 (2010). The protocol comprises three major steps: definition of a gene list from omics data, determination of statistically enriched pathways, and visualization and interpretation of the results. BMC Bioinformatics 13(Suppl. # fun = "enrichPathway". 3d, Supplementary Table 2, reporting Wilcoxon rank-sum test p-values along with BenjaminiHochberg corrected q-values). This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. To better analyze expression dynamics throughout the cell cycle, we developed a cell cycle pseudo-time inference method (Fig. 1. Nature 2019. . A novel signaling pathway impact analysis. The BCDX complex facilitates RAD51 recruitment to ssDNA. Nat. Using raw count information, Seurat objects, or SingleCellExperiment format, users can perform and visualize GSEA across individual cells. For example (Fig. Default is 0.25 pathway enrichment Description. Because the HR pathway is a key component in the G2/M checkpoint, we examined if a RAD51B expression signature of genes co-expressed with RAD51B across single KBM7 cells (Methods, Supplementary Table 4) is associated with the elevated G2/M checkpoint response we observed in near-haploid B-ALL cells. Schubert, O. T., Rost, H. L., Collins, B. C., Rosenberger, G. & Aebersold, R. Quantitative proteomics: challenges and opportunities in basic and applied research. Each color group represents an individual chromosome. Natl. These data suggest that haploid KBM7 cells display altered cell cycle expression programs in which cells show expression patterns that are mismatched with specific cell cycle stages. Given that these expression data were collected using microarrays and are thus normalized for total mRNA content per sample, they further highlight the increased dosage of RAD51B in near-haploid B-ALL. groups of cells using a Wilcoxon Rank Sum test (default), "bimod" : Likelihood-ratio test for single cell gene expression, Nat. Nature 466, 368372 (2010). Methods 11, 11381140 (2014). 15, R29 (2014). Paralllement apparaissent des socits savantes dans le domaine des sciences, de l'agriculture, de la mdecine. In addition, our gene essentiality screens have pointed to genes involved in cellular respiration and mitochondria function, including the serine hydroxymethyltransferase SHMT2 and the mitochondrial translation initiation factor MTIF2, that are also overexpressed in G2/M to M/G1 stages of the cell cycle in near-haploid cells. Surprisingly, we did not observe significant changes in total DNA damage or damage in each cell cycle stage upon RAD51B loss (Fig. Genet. c Histogram of log2-expression ratios computed from single-cell gene expression profiles in diploid and haploid KBM7 cells. By submitting a comment you agree to abide by our Terms and Community Guidelines. Forty-eight hours after doxycycline induction, cells were sorted for either protein extraction or plating growth competition assays. Correspondence to Cells were transplanted into 68-week-old male NSG mice (Jackson Laboratory) and re-harvested upon development of overt leukemia and cryopreserved as a patient-derived xenograft (PDX) line. e Co-localization of -H2AX and RAD51 foci in haploid and diploid KBM7 cells quantified using two different metrics of co-localization (schematic shown above plots), grouped by cell cycle stages. PubMed Central Cells were cultured till confluent and diluted 1:3 the night before transferring to coverslips. Protoc. An AUC value of 1 means that Genome Biol. each of the cells in cells.2). Haploid and diploidized B-ALL tumor samples populated each cluster with distinct proportions (Fig. 5d, lower panels). Both haploid and diploid cells were cultured for ~20 cell doublings before being harvested for genomic DNA extraction. Y.L. 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The following guides were used to generate vectors that allow doxycycline-inducible expression of shRNA against RAD51B as well as the E2-Crimson fluorophore in the GFP-expressing miR-30 shRNA backbone [23]: An shRNA vector against the Renilla luciferase (Ren713, guide sequence TAGATAAGCATTATAATTCCTA [24]) was also generated as control for knockdown. Nucleic Acids Res. Recovering gene interactions from single-cell data using data diffusion. Gene Set Enrichment Analysis (GSEA) is a computational method that determines whether a pre-defined set of genes (ex: those beloging to a specific GO term or KEGG pathway) shows statistically significant, concordant differences between two biological states. While hyperdiploid leukemia has a favorable outcome in general, masked hypodiploid leukemia does not show improved patient outlook and may be a source of misdiagnosis as hyperdiploid leukemia [9]. 2mL cell culture suspension was added to each well containing a coated coverslip in a 12-well plate and cultured overnight. Chapter 14 DEG GO Enrichment | Single Cell Multi-Omics Data - Bookdown Lemez P, Attarbaschi A, Bn MC, Bertrand Y, Castoldi G, Forestier E, et al. 559619), treated with DNase to expose BrdU epitope, stained with an FITC-conjugated anti-BrdU antibody as well as 7-AAD for total DNA content, followed by flow cytometry analysis. Michael T. Hemann. singleseqgset: Single-cell RNAseq gene set enrichment analysis of cells based on a model using DESeq2 which uses a negative binomial Based on the cells G2M and G1S scores, cells lie approximately in an elliptical trajectory following the order of events within the cell cycle (Fig. The same trends were observed in spleen cells. Au cours du XVIIIe sicle, l'imitation de l'Acadmie franaise, sont nes des acadmies provinciales, caractre surtout littraire. 5d, lower panels), where a subset of RAD51 proteins is recruited to damage foci to initiate repair. Biol 3, Article3 (2004). CellProfiler 3.0: next-generation image processing for biology. & Jegga, A. G. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. An AUC value of 0 also means there is perfect Option to display pathway enrichments for both negative and Cline, M. S. et al. HEK-293T cells were transfected with the above vectors (20g each in a 10-cm dish) together with the pCL-eco (12g) and pMD2.G (2.5g) vectors for retroviral packaging and VSV-g pseudotyping. 28, 495501 (2010). Prognostically relevant gene signatures of high-grade serous ovarian carcinoma. Barcoded single-cell transcriptome libraries were sequenced with 38bp paired-end reads on an Illumina on an Illumina Hi-seq 2000 instrument. 2018;36:41120. As expected, RAD51B depletion conferred a survival disadvantage in haploid KBM7 cells (Fig. Seurat - Guided Clustering Tutorial Seurat of cells using a hurdle model tailored to scRNA-seq data. Townsend EC, Murakami MA, Christodoulou A, Christie AL, Kster J, DeSouza TA, et al. 1992;10:60613. A rolling circle with an empirically determined radius of 0.5 was used to average expression in cells along the elliptical trajectory. 22, 281285 (1999). Additionally, loss of RAD51B resulted in significantly decreased co-localization of RAD51 foci with -H2AX foci in the G2/M phase of haploid but not diploid cells (Fig. Nat Biotechnol. Citation To cite this package, use RAD51 and RAD51B-mediated HR repair is mostly active in the late S to early G2 phases of the cell cycle when sister chromatids are available as recombination substrates [34]. Interestingly, the deviation in diploid-to-haploid damage foci count ratios is only significant in the G2/M phase (one-sample Students t-test p-value=0.04). This vignette shows how the ReactomeGSA package can be used to perform a pathway analysis of cell clusters in single-cell RNA-sequencing data. . An optimized lentiviral genome-wide sgRNA library [20] containing 76,441 guides targeting 19,114 human genes as well as non-targeting controls was used for transducing Cas9-expressing KBM7 cells (see above) at 1000X coverage. Rev. The Reactome pathway Knowledgebase. a Bulk gene expression ratios between diploid and haploid KBM7 cells. However, the molecular link between p53 and haploid-specific disadvantages remains unaddressed. This suggests that there is a relative increase in the frequency of unrepaired DNA breaks in haploid KBM7 cells. The protocol describes innovative visualization techniques, provides comprehensive background and troubleshooting guidelines, and uses freely available and frequently updated software, including g:Profiler, Gene Set Enrichment Analysis (GSEA), Cytoscape and EnrichmentMap. Cell 2018;174:716729.e27. 2c). A survey of best practices for RNA-seq data analysis. We explain the procedures of pathway enrichment analysis and present a. 2001;15:330818. Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. g Violin plots showing normalized RAD51B expression in B-ALL patient samples in each karyotype group. Nachman JB, Heerema NA, Sather H, Camitta B, Forestier E, Harrison CJ, et al. Bansal, V., Libiger, O., Torkamani, A. PubMed It can be seen that there is a significant reduction in the number of haploid KBM7 clones growing out upon RAD51B loss compared to control, whereas no significant difference in clone counts were observed between RAD51B knockout and control groups in diploid KBM7 cells. Pathway and network analysis of cancer genomes. Data shown in (d) and (e) are mean values of replicates.

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