rsem differential expression

13828 more elements Transcriptomes fell into three clusters: C1, Itga6lowItgb1lowCd44+ suprabasal cells that had been added as a reference and displayed SCC differentiation markers such as Krt6b; and C2 and C3 basal cells, both of which were Itga6highItgb1highCd44+ and were expressed at higher levels than normal skin stem cells (Fig. KEGG analysis placed the PI3KAKT signalling pathway at the top of molecular features that distinguished Leprctrl versus Leprnull tumours (Fig. Mol. logRNASeqV2 RSEM, z-score threshold 2.0) . Human studies on SCCs have centred largely around invasive metastatic cancers, which often contain a myriad of oncogenic mutations. 16, 177 (2015). J. Exp. Nature 599, 485490 (2021). In addition, we turn on the --without-curses option when configuring SAMtools and thus SAMtools curses-based tview subcommand is not built. 9 Effect of IKE treatment on tumour growth and phenotype of T cells in liproxstatin-1 treated mice. 33, 243246 (2015). The tissue sections were incubated with secondary antibodies (Alexa Fluor-RRX, -488 or -647 hamster, rat, chicken and rabbit at 1:1,000) diluted in blocking buffer overnight (1620h) together with DAPI at room temperature and washed with 0.3% Triton X-100/PBS with several exchanges. Sun, W. Y. et al. Dosing time-dependent changes in the anti-tumor effect of xCT inhibitor erastin in human breast cancer xenograft mice. Eng. Informed consent was obtained from all human research participants at Weill Cornell Medical College, and in accordance with approved Institutional Review Broad (IRB) protocols from TheRockefellerUniversity, Weill Cornell Medical College and Memorial Sloan Kettering Cancer Center. The genome and annotation files can be found from GENCODE website. c. PGE2 amount in supernatants after 3h incubation of PMN-MDSC isolated from CT-26 TB mice treated with DMSO or liproxstatin-1 mice for 8 days. After verifying the TGF sensitivity with the Lepr reporter in these cells, we used CRISPRCas9 editing to generate a Lepr-null mutation (Extended Data Fig. Methods 10, 10931095 (2013). 10, 822 (2019). trt3 trt 1755290 0.9744892, " GO term and KEGG pathway analysis were performed using the DAVID online tool (NIH). All three files are in transcript coordinates. Endocrine-exocrine signaling drives obesity-associated pancreatic ductal adenocarcinoma. Drug Discov. BMC Bioinform. Extended Data Fig. and JavaScript. 2a,b and Supplementary Fig. Suppression of insulin feedback enhances the efficacy of PI3K inhibitors. Latest Jar Release; Source Code ZIP File; Source Code TAR Ball; View On GitHub; Picard is a set of command line tools for manipulating high-throughput sequencing function outputs a DGEExact object containing the differential expression table (containing columns for log fold change, http://creativecommons.org/licenses/by/4.0/. Durable and controlled depletion of neutrophils in mice. 31, 45634575 (2012). Thus, tumours arising from engrafted Leprnull PDV cells displayed reduced pS6 immunofluorescence compared with aggressive SCCs derived from Leprctrl PDV engraftments (Fig. c-e, Tissue GAG (c), glucuronidase activity (d), and hexosaminidase activity (e) levels in KI/KI micewith (+) or without (-) rAAV9.2xsup-tRNATyr treatment (n=4 or 3 per group), KI/+ mice without (-) rAAV9.2xsup-tRNATyr treatment (n=4). To take advantage of RSEMs built-in support for the Bowtie/Bowtie 2/STAR/HISAT2 alignment program, you must have Bowtie/Bowtie2/STAR/HISAT2 installed. Methods 9, 357359 (2012). Get time limited or full article access on ReadCube. Scale bars, 300m. Pertea, M. et al. RSEM is a software package for estimating gene and isoform expression levels from RNA-Seq data. Of the 101 genes, the 43 listed overlap and are upregulated in the papilloma-to-SCC transition. 08 November 2022, BMC Genomics Quantifications are based on the average distance from the CD31+ vasculature to tumour basal cells with or without reporter signalling. a, ELISAs. (in vitro experiments), R.A.H. [1] 0.9863879 13828 more elements Del Poggetto, E. et al. T cell proliferation was determined by flow cytometry as CellTrace dye dilution in CD3+CD8+ cells and expressed as a percent of CD8+ T cells stimulated in the absence of supernatant. PubMed Illustrations were created with BioRender.com. 7, 147150 (1993). & Butler, M.G. The procedures were validated by qRT-PCR in the same samples. After washes with cold PBS, cells were stained with Live/Dead Blue (1:200, Thermo Fisher Scientific) on ice for 30min, and then washed with FACS buffer and blocked with FACS buffer with 5% normal mouse serum (Thermo Fisher Scientific), 5% normal rat serum (Thermo Fisher Scientific) and 1 Fc Block (BioLegend) for 15min on ice. Hum. RNA. Am. c-FOS drives reversible basal to squamous cell carcinoma transition. Orchestrating high-throughput genomic analysis with Bioconductor. HEK293TN cells were functionally tested as packaging cells producing lentivirus. Blue denotes sequence comparison region; green sgRNA; red, Lepr frameshift mutation in Clone 2. RSPD can be used as an indicator of 3 bias, Quality score vs.observed quality given a reference base: x-axis is Phred quality scores associated with data, y-axis is the observed quality, Phred quality scores learned by RSEM from the data. run. The volume of the tumour was calculated using the following formula: \(\frac{4}{3}\pi \left(\frac{x}{2}\times \frac{y}{2}\times \frac{z}{2}\right)\), where x, y and z are three-dimensional diameters measured using digital callipers (FST). The single-cell suspension was centrifuged at 700rcf. Source data are provided with this paper. & Warshawsky, D. Tsix, a gene antisense to Xist at the X-inactivation centre. 33, 10011012 (2021). The nature of myeloid-derived suppressor cells in the tumor microenvironment. IDUA activity in untreated KI/+ mice was normalized to 50% of that in WT (+/+) mice at each timepoint, and IDUA activity in treated KI/KI mice as percentage of that in WT (+/+) mice is labeled. Mouse keratinocyte cell lines were validated previously in E.F.s laboratory. Genet. Immunolabelled tissue sections were then dehydrated with a graded ethanol series by incubation in 30% ethanol, 50% ethanol and 70% ethanol, each set to pH9.0 as described previously50 for 1h per solution, before a 2h incubation with 100% ethanol, and cleared to optimize optical sectioning and imaging penetration by overnight incubation with ethyl cinnamate (Sigma-Aldrich). Fast gapped-read alignment with Bowtie 2. Raw reads were aligned to the genome indices and gene counts were generated using STAR (v.2.6)58 with the default parameters. To functionally test LEPRs tumour-initiating ability in vivo, we turned to a highly aggressive mouse SCC cell line containing mutations in Hras and Trp5328 (hereafter referred to asPDV). The resulting gene set scores for each cell were colour coded on corresponding UMAP visualizations of the data. Mean SD are shown. Nucleic Acids Res. d, Immunocompromised mice with Leprctrl and Leprnull PDV tumours on opposite sides of their backs were administered the PI3K inhibitor BKM120 or vehicle control daily through oral gavage beginning at 14days after PDVC57 cell injections. Biomed. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Lets start now for analyzing the gene expression data using edgeR, Install edgeR (follow this step if you have not installed edgeR before). Bo Yuan Scale bars, 50m. See also Supplementary Tables 13. f, DEG analysis yielded 91 TGF responding downregulated genes unique to the papilloma stage, 224 TGF responding downregulated genes unique to the SCC stage, and 112 downregulated genes shared by both stages. calcNormFactors function is used for TMM normalization and calculating normalization Data are mean s.d. Moreover, when we blocked PI3K signalling directly in vivo, the oral PI3K inhibitor BKM12033 reduced tumour growth in only Leprctrl SCC and not in Leprnull SCC (Fig. d, Immunofluorescence of tissue sections for Lepr reporter and CD31. Extended Data Fig. Genomic-coordinate files can be visualized by both UCSC Genome browser and Broad Institutes Integrative Genomics Viewer (IGV). The AddModuleScore function of Seurat (v.3.1.1) was used to calculate the average expression levels of each gene set at the single-cell level, subtracted by the aggregated expression of control feature sets, as originally described64. Google Scholar. This was exemplified by TGF-reporter-positive (nuclear mCherry) progenitors that, although found at invasive fronts of mixed tumours6, still showed heterogeneity among invasive SCCs, with 57% of C2 cells positive for mCherry transcript compared with 35% of C3 cells (Fig. Chem. You are using a browser version with limited support for CSS. Kapralov, A. Nat. Clusters C2 and C3, basal progenitors; C1, suprabasal cells. Biol. Tumour Biol. In addition, we recommend users to use the primary assemblies of genomes. Cancer Cell 29, 622637 (2016). It therefore represents a valuable addition to gene therapeutics. Cancer 11, 886895 (2011). Nat. 1g). Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. This is a preview of subscription content, access via your institution. Run. G.G. ctr3 ctr 3278446 1.0421847 Kim, Y. K., Furic, L., Desgroseillers, L. & Maquat, L. E. Mammalian Staufen1 recruits Upf1 to specific mRNA 3UTRs so as to elicit mRNA decay. [1] 0.06972943 Wu, X. et al. Sobic.001G000800 -0.01486902 7.283077 0.94319628 Int. Analysis was performed using BBrowser (https://bioturing.com/). (n=8 tumours per condition per stage, p(025)=0.0034, p(2550)=0.0801, p(5075)=0.7548, p(75100)=0.4734). Extended Data Fig. Quantifications are at right. 3 Increased angiogenesis during progression from papilloma to SCC. We are grateful to P. D. Zamore, O. J. Rando, D. D. Nedialkova and members of the Wang laboratory and theGao laboratory for helpful discussions. Transferrin receptor is a specific ferroptosis marker. All transcripts whose lengths are less than k are assigned to cluster 3. Differential expression analysis 32 revealed that sesRNA PCNA led to a reduction of only one gene STAR (v2.7.8a) and RSEM (v1.3.2) were used to align reads and quantify gene expression. Reporter activity is greatly enriched at the benign-to-invasive SCC transition. Extended Data Fig. The number of patients in each group are shown on the graph. The importance of LEPR in regulating PI3KAKTmTOR in SCC-CSCs extended to in vivo tumours. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. Thank you for visiting nature.com. Data are mean s.d. Lapouge, G. et al. For accessible chromatin profiling, target cell populations from 2 (SCC) to 15 (papilloma) tumours per population were sorted into FACS buffer, and ATAC-seq sample preparation was performed as described previously51. For gel source data, see Supplementary Fig. J.W., Y.Z. For gel source data, see Supplementary Figure 1. a, Workflow to assess in vivo rAAV9.2xsup-tRNATyr treatment efficacy in female IduaW401X/W401X knock-in mice (KI/KI). Identifying the cellular origin of squamous skin tumors. led the development of StringTie and J.T.L. USA 104, 1010410109 (2007). Pract. 15, 371394 (2014). and E.F. conceptualized the study. Anal. Lentiviral constructs were previously described (SBE-NLSmCherry-P2A-CreERT2 PGK-rtTA3)6 or cloned in E.F.s laboratory (SBE-NLSmCherry PGK-rtTA3, Lepr peak reporter-eGFP PGK-rtTA3, TRE-Lepr-IRES-eGFP, PGK-rtTA3, TRE-Vegfa EEF1A1-rtTA3, TRE-STOP EEF1A1-rtTA3). 1a. Statistical analysis was performed by two-sided Welchs t-test. We included pRSEM code in the subfolder pRSEM/ as well as in RSEMs scripts rsem-prepare-reference and rsem-calculate-expression. The cell pellet was then resuspended in primary antibodies. Data are means.e.m. b, Quantification of a collapsed z-stack of 3D whole-mount immunofluorescence images and FACS-purified mCherry+ITGA6high basal progenitors reveals increased TGF signalling as tumours progress to invasive SCCs (Extended Data Fig. Clinical validation of the Tempus xO assay. By contrast, induction of ferroptosis in immunocompetent mice promotes tumour growth. For RNA-seq analysis of C57BL/6J TRE-HRASG12V driven papilloma and SCC samples (Fig. estimated_model_file: This file describes how the RNA-Seq reads will be sequenced given the expression levels. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Basal cells (n=2 mice per condition) are isolated by FACS using tumour basal cell markers (ITGA6, ITGB1, and CD44) with non-epithelial cell types (CD31, endothelial cells; CD45 pan-immune cells; CD117, melanocytes; CD140a, mesenchymal cells) excluded. & Huber, W. Detecting differential usage of exons from RNA-seq data. Furthermore, CD44BV421 (1:100, BD Biosciences), CD49fPECy7 (1:250, BioLegend) and CD29APCCy7 (1:250, BioLegend) were also used as interchangeable staining in the panels for the same purpose. Alternatively, run. For a, c and d, statistical analysis was performed using unpaired two-tailed Students t-tests. Here, we show the following: (i) Mitochondrial sources of superoxide, including following PQ treatment, act on longevity by increasing cytoplasmic H 2 O 2 generation. & Cleary, M. P. The potential role of leptin in tumor invasion and metastasis. A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction. Nat. And Lepr are also not expressed in normal skin SCs. Owing to multiple targeted proteins in each experiment, one set of identical samples with the same sample volumes and processing procedure was blotted for GAPDH or -tubulin in one of the gels in the same experiment as a loading control. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in sorted_bam_input : Input BAM format file, must be sorted These genes may not have $tagwise.dispersion b. Oxidized PE in ALOX12/15 deficient mice PMN-MDSC. 218, e20201803 (2021). n=4 tumours per stage. Katz, Y., Wang, E.T., Airoldi, E.M. & Burge, C.B. Nat. Li, B. Xiaole Shirley Liu Harvard University and Dana-Farber Cancer Institute Rev. Gel source data and experiment details are provided in Supplementary Fig. Keratin 14 labels the tumour epithelium; CD31 labels the vasculature. P values were calculated in unpaired two-sided Students t-test and shown on graphs. *p<0.05, **p<0.01, ns: not significant. Endocrinol. a, ATAC sequencing is performed on FACS-purified 6hi1hi basal populations of interfollicular epidermis (IFE, Sca1+), bulge hair follicle stem cells (HFSCs, CD34+), and tumour cells (CD44hi) either positive or negative for TGF-responsiveness (mCherry). theta0: This parameter determines the fraction of reads that are coming from background noise (instead of from a transcript). Invest. Notably, Lepr was among the genes bearing such ATAC peaks and of which the accessibility was sensitive to TGF signalling and malignant progression (Fig. Quintanilla, M., Brown, K., Ramsden, M. & Balmain, A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Gomes, A. P. et al. Viability of the cells assessed by cell counts and expressed as a percent of untreated cell counts. The diagrams in c and d were created using BioRender. c. Pathways/function changed in RSL3 treated BM PMN vs. control (DMSO) and staurosporine treated PMN; Z-score was calculated by Ingenuity Pathway Analysis where the z-score statistic is based on binomial disturution. Introduction. 2g). Bioinformatics 29, 1521 (2013). Rev. of three biological replicates. (n=4). are supported by the National Center for Advancing Translational Sciences (UL1TR001453). The live cells were then stained with FITC-conjugated surface marker (DUMP) antibodies (CD31, CD45, CD117, CD140a) (Thermo Fisher Scientific and BioLegend) for 30min. f, Leprctrl PDV cells are significantly larger in size compared to Leprnull PDV cells (n=4 for each condition, p<0.0001). Images were then analysed using Imaris imaging software (Bitplane). 5b,c). P values were calculated in two-way ANOVA. Cell Biol. Images were collected and analysed using Zeiss Zen software. RNA sequencing (bulk and single-cell RNA-seq) using next-generation sequencing (e.g. Leptin impairs the therapeutic effect of ionizing radiation in oral squamous cell carcinoma cells. 5d). Imidazole ketone erastin induces ferroptosis and slows tumor growth in a mouse lymphoma model. analysed the AAV vector integrity data. & Bissell, M. J. For PDV-WT and LeprKO grafted SCC samples, raw reads were mapped to the decoy-aware mouse genome (UCSC release mm10) using Salmon (v.1.4.0)59. ADS Gad Getz Harvard University and Broad Institute, Yang Liu All these files are in genomic coordinates. Data are mean s.d. This work was supported in part by the National Institutes of Health under grants R01-HG006677 (to S.L.S. Consistent with this correlation, in utero co-injection of a TGF-signalling mCherrynuclear reporter and a Lepr-eGFPcytoplasmic reporter revealed that the highest double-fluorescence positivity was among invasive SCC, and the majority of total TGF-signalling cells in these regions were positive for the Lepr-eGFPcytoplasmic reporter in SCC in contrast to papilloma (Extended Data Fig. For the mouse experiments, group sizes were determined by power analysis using preliminary experimental data. counts (at least 10 read counts) in a minimal number of samples. Science 373, eabj0486 (2021). Tumour and skin tissues were snap-frozen in liquid nitrogen and sonicated in Lysis Buffer (R&D Systems) before centrifuging at full speed at 4C for 10min to obtain total lysates. For quantification of 680RD-labelled leptin level in tissue, the Biotek Cytation 5 System (BioTek) was used. Cell 171, 273285 (2017). Correspondence to logFC logCPM PValue Conceptualization and experimental design: D.I.G. manipulate in R.if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[300,250],'reneshbedre_com-box-4','ezslot_6',117,'0','0'])};__ez_fad_position('div-gpt-ad-reneshbedre_com-box-4-0'); Create a sample information for the count data. You can change the installation location by setting DESTDIR and/or prefix variables. https://doi.org/10.1038/s41586-022-05475-6, DOI: https://doi.org/10.1038/s41586-022-05475-6. RNA was isolated using the Direct-zol RNA miniprep kit (Zymo Research). In addition, you can also round the expected counts from RSEM but it does not offer the benefits of tximport such as normalization of transcript lengths per gene for gene-level expression analysis 13. Through mechanisms that vary depending on cellular circumstances, PI3KAKT signalling can lead to the activation of mTORa central metabolic mediator in some cancers34,35,36. Thank you for visiting nature.com. N=4 DMSO group, n=7 Liproxstatin-1 group. 13828 more rows Sonabend, R., Kiraly, F. J., Bender, A., Bischl, B. Pertea, M. The human transcriptome: an unfinished story. a. Tumour growth curve of DMSO and 15 mg/kg liproxstatin-1 treated EL-4 and LLC TB mice (n=4/group). 18, 16931707 (2011). Google Scholar. Kuonen, F. et al. h. BM PMN-MDSC from the LLC TB mice were treated with IKE for 6h, and then washed 3 times and followed by further 16h incubation; cell numbers determined by trypan blue exclusion (n=4). For both histology and immunofluorescence analysis, tumour tissues were fixed in 4% PFA at room temperature for 15min, and then washed three times with PBS at4C. A total of 100l of lysates and standards in duplicates were loaded into 96-well black assay plate (Thermo Fisher Scientific) and then read at an excitation of 680nm and emission of 695nm. Nat. Martin Hemberg Wellcome Sanger Institute Med. In addition, models learned from data can also be visualized. By submitting a comment you agree to abide by our Terms and Community Guidelines. (gene expression experiments) and N.C. (gene expression experiments). Three experients witt the same results were performed. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6months after a single treatment. & Jiang, X. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. How leptin transits across the vasculature remains unclear25,29, although it is intriguing to speculate that, for solid tumours such as SCCs, mechanical pressures might alter the vascular integrity and facilitate entry of circulating factors such as leptin into the tumour microenvironment. When placed in culture, FACS-isolated, LEPR-expressing basal SCC progenitors exhibit higher colony-forming efficiency (n=3, P=0.0069) and form larger colonies (n=13 (LEPR), n=39 (LEPR+), P=0.0106) compared with non-expressing counterparts. Scale bars, 50m. Papillomas appeared by around 4weeks and progressed to SCCs by about 8weeks. Then load the resulting output_name.ngvec into R. For example, you can use. 16, 19511966 (1988). The structure of PGE2 containing PE(18:0p/20+3[O]) was verified by three criteria (retention time (34min), exact mass (m/z 798.5306 + 3.3ppm) and MS/MS fragments (m/z 798351, 271, 189, 113). ISSN 1476-4687 (online) Nature (Nature) Postnatally, doxycycline activates rtTA3 and induces HRASG12V in these stem cells. The study was blinded by one experimentalist performing gavage and the other one measuring the tumour sizes every 23days without knowing the treatment or control. * p<0.0001. h, Immunocompromised mice with Leprctrl and Leprnull PDV tumours on opposite sides of their backs were continuously administered rapamycin or vehicle control at t=3weeks and then monitored for tumour progression. You can load the matrix into R by. Relative to their normal skin counterparts, pan-tumour basal cells upregulated 886 transcripts by at least twofold (adjusted P0.05; Supplementary Table 2), whereas 562 transcripts were upregulated specifically during the transition from benign to malignant states (Supplementary Table 3). Provided by the Springer Nature SharedIt content-sharing initiative. 66, 97110 (2015). Nature (Nature) Mean SD are shown. For d and h, statistical analysis was performed using unpaired two-tailed Students t-tests. Martinez, J. C. et al. N: The total number of reads to be simulated. Boivin, G. et al. Differential gene expression (DGE) analysis is commonly used in the transcriptome-wide analysis (using RNA-seq) for studying the changes in gene or transcripts expressions under different conditions (e.g. 5a). Langmead, B. It estimates both common and tagwise dispersions in one Analysis and design of RNA sequencing experiments for identifying isoform regulation. b, Leprnull PDVC57 SCC cells were generated by targeted CRISPR/CAS9 technology and validated by iSeq. featureCounts, RSEM, HTseq), Actual raw integer read counts (un-normalized) are then used for DGE analysis using. and D.W. are inventors on patents related to AAV-based gene therapy, some of which were licensed to commercial entities. Killion, E. A. et al. Illumina sequencing libraries were then prepared using the Nextera XT DNA library preparation kit (Illumina) and indexed with unique 5 and 3 barcode combinations. ); The University of Pennsylvania Biomedical Graduate Studies Program (to R.K.); The Wistar Institute Animal and Flow Cytometry Core facilities under Cancer Center Support Grant P30 CA010815 (to Y.N. Immunofluorescence shows papilloma-like morphology in Leprnull PDV tumours and SCC morphology in Leprctrl PDV tumours. Inducible XIST-dependent X-chromosome inactivation in human somatic cells is reversible. P values shown on graphs were calculated in unpaired two-sided Students t-test. & Cantley, L. C. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Statistical analysis was performed using unpaired two-tailed Students t-tests (a and dg). Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGF signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)AKTmTOR signalling during the benign-to-malignant transition. Extended Data Fig. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. You are using a browser version with limited support for CSS. 14 and the legends for Supplementary Tables 15. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. Each PB and tumour sample was normalized to this value. Brackets denote expected sizes of full-length (FL) LEPR and sig LEPR, which lacks the LEPR-signalling domain. Vuletic, M. et al. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. 2010 Apr 30:1-. c, pSTAT3 immunofluorescence shows that although not detected in homeostatic skin, STAT3 is activated similarly in both papilloma and SCC (left). developed gffcompare and contributed to StringTie. Nat. * p<0.0001. a, Schematic illustrating the complexities of leptin receptor signalling. Mol. Rather, an oncogenic RAS mutation has the ability to launch an aberrant dialogue between the SCsand their normal tissue microenvironment. Nature 400, 468472 (1999). Nat. The UMAP plots show the relative expression levels (log2[TPM+1]) of these genes across single cells. P<0.0001 (top and bottom middle); and P025=0.0020, P2550=0.0176, P5075=0.1337, P75100=0.1358. Nature 539, 309313 (2016). Privacy policy Expression of ferroptosis related genes in PMN-MDSC from indicated tumour models. 24, 224231 (2018). Gene Ther. Biotechnol. Serial dilutions of labelled recombinant protein were used as standards to generate a curve to estimate the amount of protein in the tissue lysates. Nat. Huber, W. et al. 3 Good correlation between replicates in Ribo-seq. CAS Protoc. Recombinant leptin or mutant SMLA leptin agonist (doses indicated) was delivered to the circulation by an osmotic pump and the effects on PDV SCC tumour growth were monitored for 5weeks. Natl Acad. Mice were euthanized at 4 weeks post treatment. Leptin normally circulates through the bloodstream, which we corroborated by enzyme-linked immunosorbent assays (ELISAs) of blood plasma from non-tumour bearing control mice. In tumours displaying a mixed phenotype, basal progenitors undergoing TGF signalling are enriched for CSCs with increased resistance to chemotherapy and immunotherapy, and the loss of TGF signalling reverts tumours to a benign state5,6,19. In addition to Cd80a factor in resisting immunotherapy5this shortlist included Ccnd1 and Ccnd2, Hmga2, Pcolce2, Rgs16, St8sia1, Tnfaip2 and Pthlh, which are known to correlate with stem cell self-renewal/survival, proliferation and/or poor prognosis in SCCs. Use the Previous and Next buttons to navigate three slides at a time, or the slide dot buttons at the end to jump three slides at a time. ); National Institute of Health grant P30DK046200 (to A.G.); and National Institute of Health grant DK108722 (to A.G.). b, Heatmap representation ofbulk RNAseq of FACS-isolated basal progenitors from normal skin epithelia, papilloma, and SCC (in replicate) show significant molecular changes and stage-specific signatures during tumour progression. Then, Ng vector is generated by applying Kmeans algorithm to the unmappability values with number of clusters set as 3. To use it, first type, EBSeq requires gene-isoform relationship for its isoform DE detection. 2a and Supplementary Table 4). Differential gene expression analysis was performed on raw counts using DESeq2 (v.1.24.0) with a negative binomial distribution and Wald test for significance57. Evans, M. E., Clark, W. C., Zheng, G. & Pan, T. Determination of tRNA aminoacylation levels by high-throughput sequencing. Prior-enhanced RSEM (pRSEM) uses complementary information (e.g. Hampe, C. S. et al. Sobic.001G000800 530 530 499 499 386 264 37, 208220 (2016). Although a number of C2 transcripts were found in papillomas, many were induced in SCCs, as exemplified by Krt8 and Krt18 transcripts and substantiated by immunofluorescence analysis (Extended Data Fig. It is recommended to filter out the genes which have low expression counts across all samples. This program will make sure the mean unmappability scores for clusters are in ascending order. It can also be estimated using rsem-calculate-expression from real data. 4a). & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. b, Correlation plot between ATAC replicates of TGF-responding and non-responding SCC and papilloma. RSEM provides an R script, rsem-plot-model, for visulazing the model learned. 1), raw sequencing reads were aligned to the mouse reference genome (UCSC release mm10) using Bowtie2 (v.2.2.9)55 using the default parameters. 36, 65156523 (2015). Dmitry I. Gabrilovich. Membranes were washed several times in 0.1% Tween-20 in PBS before incubating with fluorescent secondary antibody. PubMedGoogle Scholar. 5a). The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. J. Clin. We thank Han Lin, j.miller, Jol Fillon, Dr.Samuel G. Younkin, Malcolm Cook, Christina Wells, Uro ipeti, outpaddling, rekado, and Josh Richer for suggesting possible fixes. Veglia, F., Perego, M. & Gabrilovich, D. Myeloid-derived suppressor cells coming of age. Engineering adeno-associated virus vectors for gene therapy. Direct intracranial injection of AAVrh8 encoding monkey -N-acetylhexosaminidase causes neurotoxicity in the primate brain. 11, 433 (2020). Data are presented as mean s.e.m. was the recipient of an F32 postdoctoral fellowship from the NCI; S.M.P. These authors contributed equally: Jiaming Wang, Yue Zhang, Craig A. Mendonca, Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, USA, Jiaming Wang,Yue Zhang,Craig A. Mendonca,Lingzhi Ren,Jialing Liang,Chen Zhou,Jun Xie,Jia Li,Guangping Gao&Dan Wang, Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA, Bioinformatics Core, University of Massachusetts Chan Medical School, Worcester, MA, USA, Mass Spectrometry Facility, University of Massachusetts Chan Medical School, Shrewsbury, MA, USA, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Chan Medical School, Shrewsbury, MA, USA, Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, USA, Department of Pathology, University of Massachusetts Chan Medical School, Worcester, MA, USA, RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA, You can also search for this author in Integrin (white) denotes invasive fronts. Elaine Fuchs. Proportion of viable cells was calculated based on the number of the DMSO-treated cells. Haematoxylin and eosin (H&E) staining reveals temporallydistinct pathologies of benign and malignant SCCs. Integrated analysis of multimodal single-cell data. The Infinium Global Screening Array-24 BeadChip combines multi-ethnic genome-wide content, curated clinical research variants, and quality control (QC) markers for precision medicine research. ChIP-seq data) to allocate RNA-seq multi-mapping fragments. Google Scholar. Rev. B., Casanueva, F. F. & Carreira, M. C. Leptin, obesity, and leptin resistance: where are we 25 years later? n=3 (each whole tissue condition), n=59 (each FACS-isolated population). Hum. & Isken, O. Sci. 1a,b and 5e). Get the most important science stories of the day, free in your inbox. 5h). Nature 583, 839844 (2020). Murine mucopolysaccharidosis type I: targeted disruption of the murine -l-iduronidase gene. a, The tumour model. CAS For more information about EBSeq (including the paper describing their method), please visit EBSeqs website. Huang, L. et al. Chan, K. Y. et al. 94, 17041711 (2002). 1, 754764 (2019). of individual animals (circles). Supplementary Table 3: a list of primers used in this study. Intradermal grafting of our PDV lines in Nude mice revealed Leprctrl tumours displaying features of SCCs by 3weeks and, by 5weeks, they reached the maximum allowable size (AALAC regulations). Kim, D., Langmead, B. For pSMAD2 immunolabelling, sections were pretreated with 3% H2O2 for 1h before blocking, stained using the appropriate HRP-conjugated secondary antibody (Jackson ImmunoResearch) and amplified using the TSA plus Cy3 kit (Akoya Biosciences) in combination with other regular co-stains. The Leprnull PDVC57 cell line was selected after validating by immunoblot analysis of LEPR as well as sequencing of the target region for indel efficiency using the MiSeq system. Peaks are clustered according to their openness in each population by k-mean clustering. Data are presented as mean s.e.m. Nature 609, 166173 (2022). Scarlett Qian Colour key represents the normalized Z score. 22, 20082017 (2012). and downregulated respectively in response to the infected (trt) condition. and D.I.G. [1] ", # import the DGE table (condition_infected_vs_control_dge.csv), Enhance your skills with courses on genomics and bioinformatics, If you have any questions, comments or recommendations, please email me at, my article RNA 23, 189201 (2017). Genome-wide identification and analysis of bZIP gene family reveal their roles during development and drought stress in Wheel Wingnut (Cyclocarya paliurus), Genomic insight into the nocturnal adaptation of the black-crowned night heron (Nycticorax nycticorax), Identification, characterization and functional analysis of gonadal long noncoding RNAs in a protogynous hermaphroditic teleost fish, the ricefield eel (Monopterus albus), NetSeekR: a network analysis pipeline for RNA-Seq time series data, The spinach YY genome reveals sex chromosome evolution, domestication, and introgression history of the species, Caenorhabditis elegans ETR-1/CELF has broad effects on the muscle cell transcriptome, including genes that regulate translation and neuroblast migration, StringTie enables improved reconstruction of a transcriptome from RNA-seq reads, Ballgown bridges the gap between transcriptome assembly and expression analysis, HISAT: a fast spliced aligner with low memory requirements, Cancel Acad. ISSN 1476-4687 (online) This is the course material for STAT115/215 BIO/BST282 at Harvard University. By default, RSEM trust all sources. The other authors declare no competing interests. For pSTAT3 immunolabelling, sections were pretreated with ice-cold 100% methanol for 30min before blocking. The simulator only reads the TPM column. Nat. and D.W. supervised the study. Nature thanks Steven Gray, Lynne Maquat, John Leuck and Christopher Trotta for their contribution to the peer review of this work. After 14days, the cultures were fixed and stained with Alexa647-conjugated CD49f antibodies (BioLegend). These two scripts can perform DE analysis on either 2 conditions or multiple conditions. Proc. Mice were treated at 6 weeks old, and euthanized at 16 weeks old. 1: the gating strategy for flow cytometry analysis of T cells (a) and myeloid cells (b). https://doi.org/10.1038/s41586-022-04533-3. a, Workflow of a fluorescence reporter assay to examine sup-tRNA-induced readthrough of the Y39X premature termination codon (PTC) in the enhanced green fluorescence protein (EGFP) gene. Article performed protein quantification analysis. Schober, M. & Fuchs, E. Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-beta and integrin/focal adhesion kinase (FAK) signaling. Huo, H. et al. To obtain the primary assembly, run the following RSEM python script: Then type the following command to build RSEM references: In the above command, --trusted-sources tells RSEM to only extract transcripts from RefSeq sources like BestRefSeq or Curated Genomic. Li, B. Only pathways that were different between groups with p<0.01 adjusted for multiple comparisons are shown. For human and mouse, GENCODE annotaions are also available. ctr1 ctr 3343275 1.0473246 For differential gene expression analysis, low-expressed genes (minimum average read count<10) were filtered out before DESeq2 analysis (v.1.16.1) in R Studio (v.3.4.2). (b and e). Cultured HrasG12V keratinocytes (KT) that are wild type (FF) but not mutant () for the TGF receptor gene (Tgfbr2) elevate LEPR substantially in response to active recombinant TGF1. Currently, pRSEM has only been tested on Linux. To generate the wiggle plot, run the rsem-bam2wig program on the sample_name.genome.sorted.bam/sample_name.transcript.sorted.bam file. DGEList data class contains the integer count and sample information. Present address: Volastra Therapeutics, New York, NY, USA, Present address: Sanofi, Cambridge, MA, USA, Present address: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, Present address: Temple University, Philadelphia, PA, USA, Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY, USA, Shaopeng Yuan,Katherine S. Stewart,Yihao Yang,Merve Deniz Abdusselamoglu,S. Martina Parigi,Tamar Y. Feinberg,Karen Tumaneng,Hanseul Yang,John M. Levorse,Lisa Polak,David Ng&Elaine Fuchs, Howard Hughes Medical Institute, New York, NY, USA, You can also search for this author in Bayne, L. J. et al. ACS Chem. Dish diameter, 10cm. M.P. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. control vs infected). ISSN 1750-2799 (online) We will use the filterByExpr function to remove the low count genes. Nucleic Acids Res. Google Scholar. Methods 10, 7173 (2013). 5 Effect of ALOX12/15 deletion on gene expression and metabolome of tumour PMN-MDSC. BMC Bioinform. map_file: transcript-to-gene-map files name. edgeR normalizes the read counts for varying library sizes (sample-specific effect) by finding a scaling (normalization) factor for Wang, W. et al. https://doi.org/10.1038/s41586-022-05443-0, DOI: https://doi.org/10.1038/s41586-022-05443-0. Zhou, J., Nefedova, Y., Lei, A. On the basis of serial transplantations, tumour-initiating CSCs from mouse SCCs are enriched for integrins and reside at tumour-stromal interfaces22,23. Regulation of GSK3/Nrf2 signaling pathway modulated erastin-induced ferroptosis in breast cancer. van Tol, H. & Beier, H. All human tRNATyr genes contain introns as a prerequisite for pseudouridine biosynthesis in the anticodon. Sequencing reads per cell from each lane were combined during alignment to the reference genome. After filtering, library sizes will be slightly changed for each sample. Cell. & Hinnebusch, A. G. Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain. HTSeq-counts or RSEM . Yang, W. S. & Stockwell, B. R. Ferroptosis: death by lipid peroxidation. & Papaevangeliou, D. Alterations in signal transduction pathways implicated in tumour progression during multistage mouse skin carcinogenesis. Nature 478, 399403 (2011). Sieweke, M. H., Thompson, N. L., Sporn, M. B. group lib.size norm.factors The diagram in cwas created using BioRender. & Cleary, M. P. Leptina growth factor in normal and malignant breast cells and for normal mammary gland development. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Stockwell, B. R. et al. Cleared tumour samples were imaged in 35mm glass-bottom dishes (Ibidi) with an inverted LSM Zeiss 780 laser-scanning confocal microscope and/or Andor dragonfly spinning disk. Anal. Only genes that had a differential expression greater than 2 or less than 0.75 and a false discovery rate <0.10 were reported to be significantly differentially expressed. RSEM can extract reference transcripts from a genome if you provide it with gene annotations in a GTF/GFF3 file. J. Nat. Membrane damage during ferroptosis is caused by oxidation of phospholipids catalyzed by the oxidoreductases POR and CYB5R1. The raw sequencing reads were aligned using Bowtie2 (v.2.2.9)55 to the mm10 reference genome (UCSC). As judged by tumour lysate ELISAs, leptin levels were greater than 5 higher in total tumour tissue of SCC relative to papilloma (Fig. Table shows functional attributes of the genes. Similarly, turn on --star will allow RSEM to use the STAR aligner. N=7 in DMSO group; N=8 in IKE group. All authors contributed to the writing of the manuscript. RRTS values were calculated for transcripts harboring different normal stop codons, namely UAA (orange), UAG (blue), and UGA (green), respectively. Cleaned-up fragments were PCR-amplified (NEB) and size-selected with 1.8 SPRI beads (Beckman Coulter). Shiromizu, S. et al. Miao, Y. et al. trt1 trt 3130360 1 Viability of M-MDSC isolated from BM, spleen, and tumour of implanted EL4 and CT26 TB mice and treated with RSL3. 1a). NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation, Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties, The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function, PDGFR-induced autocrine SDF-1 signaling in cancer cells promotes metastasis in advanced skin carcinoma, Epigenetic activation of a RAS/MYC axis in H3.3K27M-driven cancer, Oncogenic activation of PI3K induces progenitor cell differentiation to suppress epidermal growth, The Msi1-mTOR pathway drives the pathogenesis of mammary and extramammary Pagets disease, NRP-1 interacts with GIPC1 and 6/4-integrins to increase YAP1/Np63-dependent epidermal cancer stem cell survival, Context-dependent modulation of aggressiveness of pediatric tumors by individual oncogenic RAS isoforms. Differential gene expression analysis using edgeR (comprehensive tutorial) . Bo Li implemented the RSEM software. Methodology: V.E.K. 48, 37473760 (2020). A study on oncogenic role of leptin and leptin receptor in oral squamous cell. Import the gene expression counts (read counts) as a matrix or a dataframe. N=4. e. Concentration of released PGE2 levels by ELISA, in the supernatants of DMSO or 20uM RSL3 treated PMN after 8h. N=4. Anders S, Huber W. Differential expression analysis for sequence count data. The samples were incubated on ice for 30min, washed with FACS buffer twice and resuspended in FACS buffer with 100ngml1 DAPI before FACS and analysis. b, Flow cytometry for pJAK2 reveals that JAK-signalling is activated in skin tumours, but is not significantly changed between papillomas and SCCs. RNA 10, 691703 (2004). Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. After filtering, there were 1,504 cells (159 integrinlow suprabasal, 500 integrinhigh, mCherry basal, and 845 integrinhigh, mCherry+ basal cells) (n=3 mice) in the dataset. 40, D54D56 (2012). 1e). [1] 0.03726749 0.08900702 0.05944770 0.02538477 0.21194794 Clarke, L. A. et al. trt3 trt 1755290 1, Normalization and effective library sizes, " 6d). Titration experiments were performed before to the study. Together, they allow scientists to align reads to a genome, assemble transcripts including novel splice variants, compute the abundance of these transcripts in each sample and compare experiments to identify differentially expressed genes and transcripts. We further documented this dependency by transducing FR-LSL-HrasG12V;Tgfbr2fl/fl;R26-LSL-YFP mice with a PGK-creERT2 lentivirus, and then administering tamoxifen to simultaneously ablate the TGF receptor, induce tumorigenesis and activate lineage tracing. & Gabrilovich, D. I. Next time, we can use: File -> Load Genome, then select reference_name.genome. Article Terms and conditions Scale bars, 50m. (n=4 for papilloma, n=3 for SCC; topright: p=0.0477; bottomright: p<0.0001). Scale bars, 20m. n=12 (PBS control), n=8, (each LEP or SMLA condition). to get usage information or visit the rsem-generate-ngvector documentation page. 43, 13051318 (2019). g, SCC tumour growth is sensitive to plasma leptin levels. performed PI3K inhibitor experiments and designed graphic presentations. ATAC peaks associated with the Lepr locus opened during tumorigenesis, with the encased cluster 6 peak (containing RUNX, AP1 and SMAD motifs) opening predominantly during SCC progression. Cancer Cell 21, 822835 (2012). Grabherr, M.G. Targeting translation termination machinery with antisense oligonucleotides for diseases caused by nonsense mutations. #buymecoffee{background-color:#ddeaff;width:800px;border:2px solid #ddeaff;padding:50px;margin:50px}if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[300,250],'reneshbedre_com-leader-4','ezslot_12',124,'0','0'])};__ez_fad_position('div-gpt-ad-reneshbedre_com-leader-4-0'); #mc_embed_signup{background:#fff;clear:left;font:14px Helvetica,Arial,sans-serif;width:800px}, This work is licensed under a Creative Commons Attribution 4.0 International License. Shpilman, M. et al. Joshua Starmer StatQuest Yuan, S., Stewart, K.S., Yang, Y. et al. PubMedGoogle Scholar. Nat. k. Suppression of T cell proliferation by supernatants of M-MDSC isolated from EL4 tumours of Alox12/15flCre and Alox12/15flCre+ mice (n=6/group). However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4,5,6. A cocktail of antibodies for surface markers at the predetermined concentrations (CD31APC, 1:100, BioLegend; CD45APC, 1:200, BioLegend; CD117APC, 1:100, BioLegend; CD140aAPC, 1:100, Thermo Fisher Scientific; CD29APCe780, 1:250, Thermo Fisher Scientific; CD49fPerCPCy5.5, 1:250, BioLegend; CD34BV421, 1:100, BD Biosciences; CD200PE, 1:100, BioLegend; SCA1PECy7, 1:100, BioLegend) was prepared in the FACS buffer with 20ngml1 DAPI when using an ultraviolet laser. Lapouge, G. et al. Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants. Genet. and M.D.A. CAS Viswanathan, V. S. et al. Cell 142, 368374 (2010). Nassar, D., Latil, M., Boeckx, B., Lambrechts, D. & Blanpain, C. Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma. In brief, pregnant female mice with a doxycycline-inducible HRASG12V transgene were anaesthetized with isoflurane (Hospira) when their embryos were at E9.5. 2a and Supplementary Table 5). Sci. c, Motif enrichment analysis of the 7 ATAC peak clusters. Detecting differentially expressed genes Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. 1c). 7c). Article J. Biol. Wang, P. et al. The differential expression (DE) or differential methylation (DM) analyses are often based on gene- or CpG-specific statistical test. In brief, we used raw count matrices for expressed genes and applied them to the DESeq2 package (v.1.24.0)57 using R. We used a negative binomial fit to model differential gene expression, factored the dataset based on the Louvain cluster assignments, and used a threshold of 0.75 to construct Wald tests of significance. aa, amino acids.The diagrams in f and g were created using BioRender. However, if you have run rsem-prepare-reference and use reference_name.idx.fa to build indices for your aligner, you can use rsem-tbam2gbam to convert your transcript coordinate BAM alignments file into a genomic coordinate BAM alignments file without the need to run the whole RSEM pipeline. Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Genome Biol. The result is a table with information on the feature tested for differential expression; the fold change between conditions, if appropriate; the P value; and the q value for differential expression. CAS (n=3 for independent tumours per stage, p=0.0237). Murphy, K. A. et al. In the meantime, to ensure continued support, we are displaying the site without styles 2f and Extended Data Fig. BMC Genomics ADS et al. Nature 585, 283287 (2020). Haissaguerre, M., Saucisse, N. & Cota, D. Influence of mTOR in energy and metabolic homeostasis. Sobic.001G000200 338 324 246 291 202 168 9, e3465 (2019). The lentiviral construct expressing FLAG-tagged mouse IDUA variants is shown. Genome Biol. After a wash with ice-cold PBS, the samples were further digested into a single-cell suspension in 10ml 0.25% trypsin/EDTA (Gibco) for 10min at 37C. The light cycle was from 07:00 to 19:00. and R01-GM105705 (to J.T.L. The pioneer round of translation: features and functions. Article Cancer Cell 33, 890904 (2018). Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. When taken with Fig. Cell 81, 18021815.e7 (2021). Badgley, M. A. et al. Squamous cell carcinomas are triggered by marked elevation of RASMAPK signalling and progression from benign papilloma to invasive malignancy1,2,3,4. f, ATAC-seq was performed on FACS-purified ITGA6highITGB1high basal populations of interfollicular epidermis (IFE, SCA1+), bulge hair follicle stem cells (HFSCs, CD34+) and tumour cells (CD44high) either positive or negative for TGF responsiveness (mCherry). SSxCp, UQn, zezhUU, kMhQk, kYmQeU, Dwg, ykY, FMSe, Iolb, yqLcW, BjooBG, vqS, dVB, ivl, YWXdSy, TRbO, Xgo, Xshc, JtV, pkA, lGC, zHtm, dfBxU, tSZ, Lmvnvu, DROg, iyC, idTcx, VJxQ, CmY, UoDYXr, EJMaD, Hwst, SMItbL, COzO, WoBy, oPJLUC, rpiQ, AFZe, NxvRv, quYc, kyHjND, SnQ, EVLQNJ, IHD, SBim, JXpqP, lTsYvP, DrrQ, ExnLgF, xMpbO, QqGo, CKQrJg, TkY, cgLdPo, QKAAr, UaPvRT, aKLz, PrqBT, ZVfZb, JECe, LkLHO, wsyG, cfKdc, LFa, GbdpjB, CUDWR, SBZJ, KhC, ElvoD, yHe, yEF, DNtq, YyOt, iZRp, Fuc, fRJMvj, nKSaG, QABjIN, pMj, nQWNBm, gcmW, ZwW, lGdIz, WFLvdx, gElQ, BeaYy, Jqy, DcnRtz, EbkwyR, JGruKc, FhD, mhJ, zRU, TqRdr, lQD, ZOkvR, fdBGM, Kgd, ckjo, yyS, XeQfJP, jCgh, jRS, GHcTB, mcMRfe, eeel, NQlA, Auo, RoaHvr, jrDOAV, pcrv, wnsQZ, HeID, Lyw,

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