Dear all, I use Subread in Bash for mapping and FeatureCounts for count quantification for my RNASeq data. 1. Notably, CD62L+ TPEX cells were specifically lost in the absence of MYB (Fig. FeatureCounts: featureCounts -p -T 8 -G ricegen.fa -t exon -g We next sorted MYB-deficient and control P14 TPEX cells from LCMV-Docile-infected mice and performed transcriptional profiling by RNA-seq (Extended Data Fig. The wells were washed twice using PBS. Hi, We confirmed the superior developmental properties of CD62L+ TPEX cells using single T cell transfer andfate-mapping via retrogenic colourbarcoding22,23,24,25 (Extended Data Fig. Sublong : a long-read aligner that is designed based on seed-and-vote. (ag) Congenically marked naive control (Cd4Cre) and Mybfl/flCd4Cre (Myb-cKO) P14 T cells were adoptively transferred into naive recipient mice, which were subsequently infected with LCMV-Docile. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. <== current version: 4.9.2 latest version: 4.10.1 Please update conda by running $ conda 05.fastqc 2a,b). featureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. It is considerably faster than existing methods (by an order of magnitude for gene-level summarization) and requires far less computer memory. 2022 Oct 6;14(6):plac046. Genome Biol.15, R29 (2014). <== current version: 4.9.2 latest version: 4.10.1 Please update conda by running $ conda 20, 326336 (2019). Subread package: high-performance read alignment, quantification and mutation discovery. However, this time, after running featureCounts the count output file contained only 24000 genes out of total 42000 genes in the annotation file. doi: 10.1093/nar/gkz114. 6 Development of mature CD8, Extended Data Fig. #ID After staining, samples were washed with PBS at least three times. htseq-count -t exon -i gene_id -f bam -m intersection-nonempty Q1.bam Q2.bam Q3.bam V1.bam V2.bam V3.bam riceann.gtf > result_file.txt. (c) Quantification of central memory (TCM), effector memory (TEM), CX3CR1+ and KLRG1+ cells among gp33+ CD8+ cells in Myb-cKO and control mice at indicated time points post LCMV-Armstrong infection. Thanks, I will check the bigwig on the genome browser and let you know. Nucleic Acids Res. Did you finally find out why it is so? Genomic conservation data for the human and mouse genomes were obtained from UCSC Genome Browser (https://genome.ucsc.edu). Each sample group consisted of two experimental replicates. 03. RNA-seq reads were aligned to the mouse reference genome GRCm38/mm10 using STAR (v.2.5.4)48. 1ac and Supplementary Table 1). 5l), and was further enhanced by the inhibition of PD-1 signalling in vivo (Extended Data Fig. designed the study and wrote the manuscript. eCollection 2022 Nov. Green L, Coronado-Zamora M, Rado S, Rech GE, Salces-Ortiz J, Gonzlez J. BMC Biol. Article By contrast, the larger TPEX cell cluster showed low expression of Sell but was enriched for other TPEX-cell-associated transcripts, including Icos, Xcl1, Cxcl10, Cd28 and Eomes (CD62L TPEX cells; Fig. FeatureCounts output does not contain all the genes in the annotation file, Traffic: 1235 users visited in the last hour, User Agreement and Privacy In line with this conclusion, the numbers of CD62L+ TPEX cells remained stable during PD-1 checkpoint inhibition, whereas the overall population of antigen-responsive PD-1+CD8+ T cells expanded robustly (Extended Data Fig. Does it suggest PCR amplification-based duplicated reads? Results: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. AoB Plants. mRNAcDNAssRNA-SEQTaqmRNA Epigenetic scarring of exhausted T cells hinders memory differentiation upon eliminating chronic antigenic stimulation. In brief, tags for the aligned libraries were first created using the makeTagsDirectory function within Homer then followed by peak calling using the style factor parameter with called peaks annotated to the nearest genes. 10 MYB regulates the expression of multiple genes that are critical to exhausted T cell function and maintenance. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. PMC Grassmann, S. et al. Genome Biol. (d) Box plots show frequencies and numbers of CD62L+ TPEX cells among control and PD-1-deficient P14 cells. 1e). Histone modification and transcription factors have been shown to function in the drought stress response g,h, Representative flow cytometry plots (g) and box plot (h) showing the fold change of frequencies of splenicpolyclonal PD1+CD8+ T cells in anti-PD-L1-treated versus PBS-treated mice. SRR3286802 Careers. Immunity 46, 7891 (2017). Id3GFP P14 cells were adoptively transferred into naive mice, which were subsequently inoculated with LCMV-Docile, which causes chronic infection (Fig. Dots in graphs represent individual mice; box plots indicate range, interquartile and median. A paired or unpaired Students t-test (two-tailed), Welchs t-test, MannWhitney U test or one-way ANOVA was used to assess significance. P values are from MannWhitney tests (g) and two-tailed unpaired t-test (i); P>0.05, not significant (n.s.). Smyth, G. K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. 3a). 20Question and Answer1 Samples were incubated with antibodies against B220 (RA3-6B2, eBioscience), CD3 (17A2, eBioscience) and F4/80 (BM8, Biolegend) diluted in 5% NGS for 2h at room temperature in the dark. 4ce). Splenic P14 T cells of R2 mice were analysed at day 28 post re-transfer. and S.J. Nature 571, 211218 (2019). 11ad and Supplementary Table 3). Splenic P14 TPEX cells were sorted at day 7 post-infection and processed for bulk RNA-seq. c, Heat map showing the expression of all identified cluster signature transcripts. Further information on research design is available in theNature Research Reporting Summary linked to this article. Haematopoietic stem cells (HSCs) were then sorted as live CD3CD19Sca-1+ cells and cultured at 37C in cDMEM(DMEM (Life Technologies), supplemented with 10%FCS, 0.025% l-glutamine, 0.1% HEPES, 0.1% gentamycin and 1% penicillin/streptomycin), supplemented with 20ngml1 mouse IL-3, 50ngml1 mouse IL-6 and 50ngml1 mouse SCF, for three to four days in tissue-culture-treated 48-well-plates. J. Exp. Sci. These authors contributed equally: Carlson Tsui, Lorenz Kretschmer, Svenja Rapelius, These authors jointly supervised this work: Veit R. Buchholz, Axel Kallies, Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia, Carlson Tsui,Sarah S. Gabriel,Daniel T. Utzschneider,Teisha Mason,Santiago Valle Torres&Axel Kallies, Institute for Medical Microbiology, Immunology and Hygiene, School of Medicine, Technical University of Munich (TUM), Munich, Germany, Lorenz Kretschmer,Svenja Rapelius,Sebastian Jarosch,Justin Leube&Veit R. Buchholz, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia, David Chisanga,Yang Liao,Stephen A. Wilcox,Stephen L. Nutt&Wei Shi, Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia, School of Cancer Medicine, La Trobe University, Melbourne, Victoria, Australia, Wrzburg Institute of Systems Immunology, Max Planck Research Group, Julius-Maximilians-Universitt Wrzburg, Wrzburg, Germany, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia, Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich (TUM), Freising, Germany, School of Computing and Information Systems, University of Melbourne, Melbourne, Victoria, Australia, You can also search for this author in Genes were called differentially expressed if they achieved an FDR of 15% or less. (h) Size of single-T-cell-derived progenies and frequencies of CD62L+, KLRG1+ and (i) PD-1+ cells therein. featureCountscountscpmtpm setwd("~/Desktop")# countdata<-read.table(" APP IT https://www.jianshu.com/p/e8cd62ba14fe PubMedGoogle Scholar. DNA(1)RNA(RNA-seq)(qPCR)[2-4] (m) Schematic of the experimental set-up. Ouzhan Klekci M, Hon WK, Shah R, Scott Vitter J, Xu B. BMC Genomics. (h) Schematic of the experimental set-up. USA 102, 1554515550 (2005). Splenic P14 T cells of R2 mice were analysed at day 14 post re-transfer. 19). 02. Furthermore, the superior proliferative and developmental potential of TSLEX cells makes them prime targets of adoptive T cell transfer and chimeric antigen receptor (CAR) T cell therapies. Nature 571, 265 (2019). (h) Flow cytometry plots and quantification showing the frequencies of PD-1+ cells among splenic CD8+ T cells. 10c and Supplementary Table 2). sampleIDread. Installation. Each cell staining reaction was preceded by a 10-min incubation with purified anti-mouse CD16/32 Ab (FcgRII/III block; 2.4G2) and (fixable) viability dye (Thermo Fisher Scientific). b, Violin plots showing normalized expression of Sell and Myb. Notably, single CD62L+ TPEX cells exhibited self-renewal and multipotent repopulation capacity, akin to single naive T cells (Extended Data Fig. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Histone modification and transcription factors have been shown to function in the drought stress response 10hj and Supplementary Table 2). source activate python3 They are completely missing from the output. 2022 Dec 8. doi: 10.1038/s41591-022-02107-4. In brief, bone marrow was collected from congenically marked (CD45.1+ or CD90.1+) P14 donor mice and stained with Ly6A/E (Sca-1), anti-mouse CD3 and CD19 antibodies, together with propidium iodide for live or dead discrimination. linux100101subread (rnaseq) root 12:08:22 ~ $ conda install -y subread Collecting package metadata (current_repodata.json): done Solving environment: done ==> WARNING: A newer version of conda exists. 20, 337349 (2019). Immunol. This state of restrained functionalitywhich is referred to as T cell exhaustion1,2is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1 exhausted effector T cells3,4,5,6. Thus, MYB represents a transcriptional checkpoint that instructs the differentiation and function of CD8+ T cells in response to severe or chronic infection. (d) Flow cytometry plots depicting combined staining of CD45.1 and Thy1.1 with KLRG1 (upper row), or CD62L with PD-1 (lower row) for three progenies derived from adoptively transferred single naive P14 cells (grey: endogenous CD4CD19 cells). WebfeatureCountsubreadsubreadsubreadR featureCounts Feature: exon; featureCounts: a ultrafast and accurate read summarization program. These results indicate that PD-1 signalling does not affect the development or maintenance of CD62L+ TPEX cells but limits their differentiation into CD62L TPEX and TEX cells. (ij) Flow cytometry plots (i) and quantification (j) showing kinetics of splenic polyclonal PD-1+ TPEX cells among Myb-cKO and control CD8+ T cells after infection. featureCountssubreadsubjuncsubread featureCounts 1.BAM/ SAM bambam This work was supported by the National Health and Medical Research Council of Australia (NHMRC) Research Fellowship (to A.K.) (q) Flow cytometry plots and quantification show the frequencies of Ly108+ and CD62L+ cells among splenic antigen-specific (gp33+) T cells in wild-type mice at day 8 post LCMV-Docile or LCMV-Armstrong infection. New filters are being implemented. Results: Google Scholar. CD45.1 or CD45.2 mice were obtained from the Australian Resources Centre or were purchased from Envigoat 68 weeks of age. Frebel, H. et al. All steps were performed using RPT filter tips (Starlab) and LoBind tubes (Sigma). Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells. For secondary single-cell re-transfers, anti-mouse CD45.1, anti-mouse CD62L and anti-mouse Ly108 were used, together with the eBioscience Fixable Viability Dye eFluor 780 or propidium iodide for live or dead discrimination. We acknowledge the Melbourne Cytometry Platform for provision of flow cytometry services and the NIH Tetramer Facility for providing tetramers. (gl) Congenically marked naive P14 T cells were transferred into primary recipient mice (R1), which were then infected with LCMV-Cl13. It includes Subread aligner, Subjunc exon-exon junction detector and featureCounts read summarization program. Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. 8hj). Of note, maximum cell numbers attainable for each subset were transferred to allow for reliable evaluation of phenotypic diversification in expanded progenies. 12, 9921001 (2011). (c) Volcano plots highlighting genes differentially expressed (FDR<0.15) between control CD62L+ TPEX and CD62L TPEX cells. Splenic CD8+ T cells were analysed at 31 dpi. This table will then be used to perform statistical analysis and find differentially expressed genes. Flow cytometry plots show the expression of PD-1, Ly108 and CD62L in splenic P14 T cells. 11f,g), contained more KLRG1+ effector cells and provided significantly enhanced viral control compared to the other exhausted T cell subsets (Fig. (a) Schematic of the experimental set-up. To test this model experimentally, we sorted CD62L+ TPEX, CD62L TPEX and TEX P14 cells on day 28 after infection with LCMV-Cl13, separately re-transferred them into congenically marked infection-matched hosts and analysed three weeks later (Fig. As a result, mice that lacked MYB in their T cells succumbed to chronic but not acute viral infection, highlighting that T cell exhaustion is an essential adaptation to chronic infection. 10d,e). PubMed Central 2022(PPT & ppt featureCountscountscpmtpm setwd("~/Desktop")# countdata<-read.table(" APP IT To do this we must summarize the reads using featureCounts or any other read summarizer tool, and produce a table of genes by samples with raw sequence abundances. featureCounts -p -T 8 -G ricegen.fa -t exon -g gene_id -a riceann.gtf -F GTF -M -O --fraction -o STRcounts.txt Q1.bam Q2.bam Q3.bam V1.bam V2.bam V3.bam, HTSeq-counts: , 555: The smaller of these clusters was characterized by high expression of transcripts that are typically associated with naive or central memory T cells, including Sell (encoding CD62L), Ccr7, S1pr1, Lef1, Satb1 and Bach2 (referred to as CD62L+ TPEX cells; Fig. Nucleic Acids Res. subtype_MethyLevel, LiAiMiAi: (d) Flow cytometry plots showing the expression of Id3-GFP, TCF1 and CD62L among splenic P14 T cells at 7 and 21 dpi. (i) Flow cytometry plots and box plots showing the frequencies of CX3CR1+ and CD101+ cells among recovered TEX cells derived from the indicated re-transferred TPEX subsets at day 28 post re-transfer. 10, 468 (2019). Provided by the Springer Nature SharedIt content-sharing initiative. Graphs and statistical analyses were done with Prism 7 (GraphPad Software). 11, R14 (2010). GMFI, geometric mean fluorescence intensity. Subread package: high-performance read alignment, quantification and mutation discovery. Subread package: high-performance read alignment, quantification and mutation discovery. Data are representative of two independent experiments. For further analysis in this study, datasets GSM5135522 and GSM5135523 (ref. LinuxCondaCondamacAapp Storecondacondacondacondaconda In line with the epigenetic imprint of exhaustion26,27,28, progeny derived from single CD62L+ TPEX cells maintained high levels of PD-1 expression compared to their naive-derived counterparts (Extended Data Fig. https://doi.org/10.1038/s41586-022-05105-1, DOI: https://doi.org/10.1038/s41586-022-05105-1. For isolating naive CD8+ or transgenic P14 T cells, the mouse CD8+ T cell enrichment kit (Miltenyi Biotech) was used, or cells were sorted as live CD8+CD44low cells. Symbols and error bars represent mean and s.e.m., respectively. subreadfeatureCountscounts_id.txt DESeq2limmaDESeq2 First step: f, Schematic of the experimental set-up. 20, 651668 (2020). Compared with CD62L TPEX and TEX cells, CD62L+ TPEX cells showed a superior repopulation capacity (Fig. Consistent with our observations in non-chimeric mice, MYB-deficient antigen-specific CD8+ T cells proliferated more and exhibited increased expression of effector molecules compared to controls (Extended Data Fig. The library was sequenced with 20,000 reads per cell. Google Scholar. 209, 24852499 (2012). Splenic P14 T cells of R3 mice were analysed at day 14 post re-transfer. WebfeatureCounts 1. 8kn). Finally, our results show that two central but seemingly unrelated properties of exhausted T cellslimited function and longevityare intimately linked by a single transcription factor MYB; this is a notable example of evolutionary parsimony, which ensures ongoing T cell immunity during chronic infection while preventing collateral damage to the host. Med. To study the role of MYB in CD8+ T cells during viral infection, we infected Mybfl/flCd4Cre mice35 (which lack MYB specifically in T cells) and Mybfl/fl (control) littermates with LCMV-Docile or LCMV-Armstrong (Fig. Please also post featurecounts summary, if possible. (p) Naive MybGFP and wild-type (non-reporter, control) CD8+ T cells were stimulated and cultured in vitro using plate-bound anti-CD3. PanGen mPDAC MS proteomics 3c). Khan, O. et al. A comparison of MYB-deficient and control CD62L TPEX cells identified 580 differentially expressed genes (Supplementary Table 2), including genes that encode molecules related to T cell exhaustion and TPEX cell identity (Lef1, Eomes, Ctla2a, Irf4, Ikzf2, Nt5e and Cd160), cell-cycle regulation and stem cell renewal (E2f1, Rbl2, Kif14, Cdc25b, Bmp7 and Wnt3) (Fig. (b) Flow cytometry plots showing the sorting strategy. Functional annotation of our scRNA-seq data identified Myb, encoding the transcription factor MYB(also called c-Myb), as specifically enriched among CD62L+ TPEX cells (Fig. Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. featureCountssubreadsubjuncsubread featureCounts 1.BAM/ SAM bambam 12b). (j) RNA velocity analysis showing developmental trajectories of TPEX cells, together with the expression of Tcf7 (left) and Sell (right). WebConda. Splenic P14 T cells were analysed at 7 dpi. Comparative analysis of algorithms for next-generation sequencing read alignment. Tsui, C., Kretschmer, L., Rapelius, S. et al. Immunol. Single-cell suspensions were obtained by mashing total spleens, lymph nodes or bone marrow through a 70-m nylon cell strainer (BD). (ij) Flow cytometry plots (i) and quantification (j) showing expression of CD62L among polyclonal TPEX cells (Ly108hiTIM-3lo). For the binned clustering analysis, the genome was split into bins of 3000 bp and for each bin, in each sample, reads were counted using the summarizeOverlaps function from the R package 2011 Oct 15;27(20):2790-6. doi: 10.1093/bioinformatics/btr477. CAS Dobin, A. et al. Sublong : a long-read aligner that is designed based on seed-and-vote. 7p). 2g and Extended Data Fig. Before k, Schematic of the experimental set-up. Read counts per gene locus were obtained with htseq-count (v.0.11.4)49. Batch effects were identified using sva (v.3.34.0) and subsequently modelled in the DESeq2 design formula. Length -g # meta-featuregene_id Rev. PubMed Cells are colour-coded on the basis of pseudotime prediction. 2m,n and Extended Data Fig. Epub 2012 Oct 9. Libraries were quantified with the Qubit dsDNA HS Assay Kit (Life Technologies Q32851). To identify factors that promote the self-renewal and multipotency of TPEX cells, we performed single-cell RNA sequencing (scRNA-seq) of TPEX-cell-enriched (PD-1+TIM-3) CD8+ T cells sorted at 30days post-infection (dpi) from mice chronically infected with lymphocytic choriomeningitis virus (LCMV) clone-13 (Cl13). CD62L+ TPEX cells also gave rise to more CX3CR1+ TEX cells (Extended Data Fig. CD62L+ TPEX cells were enriched in the spleen and lymph nodes, but largely absent from the blood, bone marrow and liver (Extended Data Fig. d, Congenically marked naive P14 cells were transferred into recipient mice, which were subsequently infected with LCMV-Docile and analysed at 21dpi. (kp) Congenically marked naive Nur77-GFP reporter P14 T cells were transferred into naive (km) or CD4-T-cell-depleted (np) recipient mice, which were subsequently infected with LCMV-Cl13. WebSubread is a read aligner. featureCounts(subread) v2.0.1 featureCounts SubreadsubreadSNPSubread The https:// ensures that you are connecting to the For LCMV-Armstrong infection, frozen stocks were diluted in PBS and 2105 PFU were injected intraperitoneally. (b) Box plot showing PD-1 expression of transferred P14 cells at 8 dpi. (b) Flow cytometry plots and quantification of IFN+ cells among Myb-cKO and control CD8+ T cells after peptide restimulation in vitro at 8 dpi. 1ac). Cluster-specific genes were identified with the FindAllMarker function in Seurat with parameters min.pct=0.25, logfc.threshold=0.25. 3. Horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. Bookshelf Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 3d and Extended Data Fig. FDM; FineSplice Enhanced splice junction detection and estimation from RNA-Seq data. PubMed Central https://ibb.co/SmD3cK2. 2e,f and Extended Data Fig. 43, e47 (2015). ac, Congenically marked Mybfl/flCd4Cre (Myb-cKO) and Cd4Cre (control) P14 T cells were adoptively transferred into naive recipient mice, which were then infected with LCMV-Docile. Extended Data Fig. (c) Quantification of GZMB expression among gp33+ cells of the indicated genotypes. 14, 768774 (2014). Veit R. Buchholz or Axel Kallies. (f) Spleen size (left) and haematoxylin and eosin staining of sections showing infiltration of immune cells (arrows) in livers (middle) and lungs (right) in Myb-cKO and control mice at 8 dpi. Google Scholar. The indicated subsets were then sorted according to their expression profile of CD62L and Ly108 (note: the anti-mouse CD62L antibody was titrated to a dilution that precludes functional blocking of the molecule). Google Scholar. Nat. Dots in graph represent individual mice; box plots indicate range, interquartile and median. BMC Genomics. Immunol. (h). Appl. (h, i) Congenically marked naive P14 T cells were transferred into primary recipient mice (R1), which were then infected with LCMV-Docile. (b) Enrichment of Ly108+CD69+ (TEX prog1) and Ly108+CD69 (TEX prog2) signatures29 at single-cell and cluster levels. 22, 10201029 (2021). Installation. After several weeks, colour-barcoded naive (CD8+CD44low) P14 cells were sorted from the peripheral blood of retrogenic mice and transferred into C57BL/6 recipients. Mansour, M. R. et al. Online ahead of print. (a) Schematic of the experimental set-up for the naive P14 single-cell transfer. Nat. Results: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. (j) Image and box plot showing the size and weights of spleens in untreated and CD8+ T-cell-depleted Myb-cKO mice at day 8 post LCMV-Docile infection. (f) Schematic of the experimental set-up. Proliferating transitory T cells with an effector-like transcriptional signature emerge from PD-1+ stem-like CD8+ T cells during chronic infection. 10f,g). 12c), the frequenciesbut not the numbersof CD62L+ TPEX cells were markedly decreased compared to control P14 T cells (Extended Data Fig. Source data are provided with this paper. Clipboard, Search History, and several other advanced features are temporarily unavailable. Other intracellular staining was performed with the Foxp3 transcription factor staining kit (eBioscience) and the following antibodies: TCF1 (C63D9, Cell Signaling), GZMB (MHGB04, Thermo Fisher Scientific) and Ki67 (FM264G, BD). SCALCE: boosting sequence compression algorithms using locally consistent encoding. MYB also mediates functional exhaustion during chronic infection by restricting the initial expansion and effector function of antigen-responsive CD8+ effector T cells. MYB has important roles in the self-renewal of haematopoietic stem cells and cancer cells30, T cell leukaemia31 and CD8+ memory T cells32,33. To characterize the dynamics of Myb expression in chronic infection, we infected MybGFP reporter mice34 with LCMV-Docile (Fig. 08.sam to bamreads ); the European Research Council (starting grant 949719 SCIMAP to V.R.B. sharing sensitive information, make sure youre on a federal 2022(PPT & ppt Article featureCounts(subread) v2.0.1 featureCounts SubreadsubreadSNPSubread subreadfeatureCountscounts_id.txt DESeq2limmaDESeq2 First step: (f) Schematic of the experimental set-up. (f) Flow cytometry plots of progenies recovered at 8 dpi. Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8+ central memory T cells. It can be used to count both RNA-seq and genomic If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. (b) Representative flow cytometry plots showing the sorting strategy and post-sort purities. 1ik). These characteristics were maintained even after repetitive adoptive transfers (Extended Data Fig. Zander, R. et al. Cell 177, 18881902 (2019). If you have a new idea or new request, please file an issue. P values are from MannWhitney tests (hi); P>0.05, not significant (n.s.). Raw gene counts were quantified using the featureCounts program of subread v2.0.1. Red blood cells were lysed with a hypotonic ammonium chloride-potassium bicarbonate (ACK) or ammonium chloride-Tris (ACT) buffer. Splenic P14 T cells were analysed at day 8 post LCMV-Armstrong infection. (de) Mixed bone marrow chimeric mice containing congenically marked Myb-cKO and control CD8+ T cells were infected with LCMV-Docile. Nat. FeatureCounts: featureCounts -p -T 8 -G ricegen.fa -t exon -g Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. There were no major differences in cytokine production between the two TPEX subsets, but IFN+ cells were enriched among CD62L+ TPEX cells (Extended Data Fig. 10h,j). In addition, naive P14 cells were included for the analysis. (l) Schematic of the experimental set-up. government site. ); the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; SFB-TRR 338/1 2021452881907, SFB 1054210592381 to V.R.B. 63) and then normalized to TPM values. The sequencing data shown in Extended Data Fig. It is considerably faster than existing methods (by an order of magnitude for Thus, exhausted CD8+ T cells constitute a dynamic network of phenotypically and functionally distinct populations that ultimately depend on the functionality of TPEX cells. Data are representative of two independent experiments (d). 19) were combined with another publicly available scRNA-seq dataset of mouse exhausted CD8+ T cells, accessed from GSE122712 (ref. 3 Capacity for self-renewal and multipotent differentiation is restricted to the CD62L, Extended Data Fig. and A.K. 50,52). Normalization and differential gene expression analysis was performed using DESeq2 (v.1.26.0). 2011;12 Suppl 2(Suppl 2):S7. Mounted samples were imaged using a Nikon SMZ1270 Stereo Microscope. Dear all, I use Subread in Bash for mapping and FeatureCounts for count quantification for my RNASeq data. Most of the sequencing data generated for this study have been deposited in the NCBI GEO database with accession number GSE188526. The sequencing data are available at the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE205608. Utzschneider, D. T. et al. Heinz, S. et al. Dots in graph represent individual mice; box plots indicate minimum and maximum values (whiskers), interquartile range (box limits) and median (centre line). Genet. Mice were left to recover for six to eight weeks before further experiments. Raw reads counts were assigned to Ensembl 75 genes using Subread v1.4.6 38, normalized for library depth and gene size (RPKM), and log 10-transformed. Uncovering the molecular mechanisms of drought stress response has been one of hot research topics worldwide. fh, Mixed bone marrow chimeric mice containing congenically marked Myb-cKO and Cd4Cre (control) T cells, infected with LCMV-Docile, were treated with anti-PD-L1 on days 33, 36, 39, 42 and 45 and analysed at 49dpi. 5k). Thank you for visiting nature.com. (an) Naive CD45.1 mice were lethally irradiated and reconstituted using a mixture of Mybfl/flCd4Cre (Myb-cKO) and Cd4Cre or littermate Mybfl/fl control (Ctrl) bone marrow. RNA-seq reads were aligned to the mouse reference genome GRCm38/mm10 using the Subread aligner (Rsubread v.2.2.6) 50. J. Biochem. 1. Chimeric mice were subsequently infected with LCMV-Docile and analysed at the indicated time points after infection. Annu. Combined analysis of our data and publicly available scRNA-seq datasets11,19 (Fig. 4, 11841191 (2009). 04.sra to fastq 4f. Science 340, 630635 (2013). c, MybGFP reporter mice were infected with LCMV-Docile and splenic CD8+ T cells were analysed at the indicated time points after infection. TPEX cells also mediate the response to therapeutic checkpoint inhibition3,5,10,11, which can reinvigorate exhausted CD8+ T cell responses and has revolutionized cancer therapy12. 2019 May 7;47(8):e47. GFOLD Generalized fold change for table(duplicated(lnc$id)) Reads were aligned to the genome using star v2.6.0c and reads in genes were counted with featureCounts (subread v1.6.2) and parameter -s 0. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. ce, Congenically marked naive P14 T cells were transferred into CD4-depleted R1 mice, which were subsequently infected with LCMV-Cl13. fk, Congenically marked naive P14 T cells were transferred into primary recipient (R1) mice, which were then infected with LCMV-Cl13. i,j, Flow cytometry plots and quantification showing the expression of IFN and TNF after gp33 peptide restimulation (i) and the frequencies of CD62L+ TPEX cells (j). featureCounts(subread) v2.0.1 featureCounts SubreadsubreadSNPSubread To directly test the role of CD62L+ TPEX cells incheckpoint blockade, we performed adoptive transfer experiments in the context of therapeutic PD-1 inhibition (Fig. Installation. 1 Isolation of polyclonal exhausted T cells for scRNA-seq and phenotypic characterization of CD62L, Extended Data Fig. MYB-deficient TPEX and TEX cells expressed significantly higher levels of IFN and TNF, whereas TEX cells also expressed more granzyme B and underwent increased proliferation (measured by the expression of Ki67) compared to controls (Fig. (b) Quantifications showing cytokine production of each subset after restimulation. PubMed 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. Epub 2011 Jul 27. The sequencing data are available at the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE205608. feature 2. 20Question and Answer1 This table will then be used to perform statistical analysis and find differentially expressed genes. To test the functional potential of MYB-dependent CD62L+ TPEX cells, we separately transferred CD62L+ and CD62L TPEX cells as well as TEX cells into either wild-type (Extended Data Fig. Nature 560, 494498 (2018). The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells. Afterwards, the single cells were encapsulated into droplets with the ChromiumTM Controller (10X Genomics) and processed following the manufacturers specifications. A.K. RNA-seq (e) Flow cytometry plots and quantifications showing the production of cytokines as indicated from Myb-cKO and control P14 T cells after gp33 peptide restimulation at 8 dpi. -F # GTF Genet. The indicated subsets of P14 T cells were sorted from R2 mice at 35 dpi and 1~3103 cells were re-transferred to infection-matched tertiary recipient mice (R3). Abdel-Hakeem, M. S. et al. linux100101subread (rnaseq) root 12:08:22 ~ $ conda install -y subread Collecting package metadata (current_repodata.json): done Solving environment: done ==> WARNING: A newer version of conda exists. P values are from two-tailed unpaired t-tests (c, l, or) and MannWhitney tests (f); P>0.05, not significant (n.s.). Motivation: Gene-level read counts were obtained by running featureCounts51, a read count summarization program within the Rsubread package52 and the inbuilt Rsubread annotation that is a modified version of the NCBI RefSeq mouse (mm10) genome annotation build 38.1. (o) Histograms showing Myb-GFP expression of gp33+ and naive (gated on CD62L+CD44) CD8+ T cells in the same mice. (a) Schematic of the experimental set-up. 20Question and Answer1 In mice, TPEX cells are defined by the co-expression of PD-1, the transcriptional regulators TCF1 and ID3 and the surface molecules CXCR5 and Ly108. To obtain Perhaps try to run something like AGAT on it , it may be able fix this. featureCounts: a ultrafast and accurate read summarization program. Frumento, G. et al. logFC, log-transformed fold change. gffread -E riceann.gff3 -T -o riceann.gtf, Subread mapping: 2hj, TPEX cells were sorted as live CD8+PD1+CD45.1+Tcf7GFP+ cells from the spleens of chronically infected mice (LCMV-Cl13, 28dpi) using a MoFlo Astrios cell sorter (Beckman Coulter) and processed using the 10X Genomics technology, according to the manufacturers protocol (Chromium Single Cell 3 GEM v3 kit). subread, RNA-seq & WebThe threshold for low complexity filter can be specified by -Y or --complexity_threshold.It's range should be 0~100, and its default value is 30, which means 30% complexity is required.. Other filter. Statistical methods to predetermine sample size were not used. Data analysis CUT&Tag sample clustering. Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. featureCounts is a highly efficient general-purpose read summarization program that counts mapped reads for genomic features such as genes, exons, promoter, gene bodies, genomic bins and chromosomal locations. Disclaimer, National Library of Medicine CAS (b) Flow cytometry plots showing expression of GFP and YFP (left) or BFP/CFP and CFP/T-Sap (right) in peripheral blood of retrogenic P14 donor mice (pre-gated on CD8+CD44loCD45.1+). Unfortunately not. Rev. Mol. TCF-1-centered transcriptional network drives an effector versus exhausted CD8 T cell-fate decision. ; APP2001719 to I.A.P. conda install -c bioconda subread --yes Command Finally, the proportions of CD62L+ antigen-specific CD8+ T cells were 10-fold higher in LCMV-Docile versus LCMV-Armstrong infection (Extended Data Fig. Many of the genes that were dysregulated in the absence of MYB, including Tcf7, Kit, Slamf6, Lef1, Klf2, S1pr1, Icos, E2f1, Gzma, Gzmc and Myb itself, contained MYB-binding regions in human T cells38 (Supplementary Table 3), which were conserved and aligned with open chromatin regions in mouse exhausted T cells13 (Fig. T cell exhaustion is an important physiological adaptation to continuous antigen stimulation in chronic infection and cancer, and although it protects against excessive immune-mediated tissue damage, it also contributes to viral or tumour persistence1,2,4,7. WebfeatureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. 69, 301318 (2018). d, Flow cytometry plots and quantification show the frequencies of KIT+ cells among control and Myb-cKO TPEX P14 T cells at day 8 after infection with LCMV-Docile (gated on TPEX cells). 63) and then normalized to TPM values. WebThe threshold for low complexity filter can be specified by -Y or --complexity_threshold.It's range should be 0~100, and its default value is 30, which means 30% complexity is required.. Other filter. RNAseq These findings not only advance our understanding of the mechanisms of T cell re-invigoration in the context of checkpoint inhibition, but also emphasize the need for new therapeutic strategies that target TSLEX cells to harness the full potential of T cell-mediated immunotherapy. Here is the FeatureCounts output screenshot image for the 6 bam files as input in the code above: By submitting a comment you agree to abide by our Terms and Community Guidelines. PFU, plaque-forming units. (l) Histograms (filled) show Myb-GFP expression of gp33+ CD8+ T cells in mice infected with LCMV-Docile (top) and LCMV-Armstrong (bottom). For liver samples, lymphocytes were obtained by density gradient centrifugation. Intratumoral Tcf1+PD-1+CD8+ T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Cell Biol. In the meantime, to ensure continued support, we are displaying the site without styles Get the most important science stories of the day, free in your inbox. Accessibility (l) Flow cytometry plots and quantifications showing CX3CR1 and CD101 expression in Myb-cKO and control TEX cells at the indicated time points after infection. Article P values are from two-tailed unpaired t-tests (c,f,hj) and MannWhitney tests (ln). Nat. Nssing, S. et al. (s) Quantification showing the absolute numbers of splenic CD8+, gp33+, CD62L+ TPEX, CD62L TPEX and TEX cells in Myb-cKO and control mice at day 8 post LCMV-Docile infection. Immunol. Proc. Immunol. Data are representative of all analysed mice (e,g), two (c,f,i,j,ln) or three independent experiments (h). 1d and Extended Data Fig. conda activate python3 Peer reviewer reports are available. How is read coverage for missing genes per exon (for eg take one and check)? (a) Schematic of the experimental set-up. kn, Mixed bone marrow chimeric mice containing Myb-cKO and Cd4Cre control T cells were infected with LCMV-Docile and analysed at the indicated time points. PubMed Central 38, 14081414 (2020). Since I have little time left for this work, I shifted my analysis from SubRead to Salmon based reference transcriptome analysis where I do not need the FeatureCounts or HTSeqCount. Nat Med. index<-duplicated(lnc$id) Nucleic Acids Res. Yes, the names are the same. Thompson RC, Simons NW, Wilkins L, Cheng E, Del Valle DM, Hoffman GE, Cervia C, Fennessy B, Mouskas K, Francoeur NJ, Johnson JS, Lepow L, Le Berichel J, Chang C, Beckmann AG, Wang YC, Nie K, Zaki N, Tuballes K, Barcessat V, Cedillo MA, Yuan D, Huckins L, Roussos P, Marron TU; Mount Sinai COVID-19 Biobank Team, Glicksberg BS, Nadkarni G, Heath JR, Gonzalez-Kozlova E, Boyman O, Kim-Schulze S, Sebra R, Merad M, Gnjatic S, Schadt EE, Charney AW, Beckmann ND. We also observed increased expression of Kitencoding KIT, which is involved in haematopoiesis and T cell activation36,37in MYB-deficient versus wild-type TPEX cells (Fig. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. featureCounts implements highly efficient chromosome hashing and feature blocking techniques. Notably, CD62L+ TPEX cells were transcriptionally distinct from both naive and memory T cells derived from acute LCMV infection (Extended Data Fig. Symbols and error bars in (l) represent mean and s.e.m., respectively Data are representative of two independent experiments (e, g, k, l). 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