He, R. et al. 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 22, 10201029 (2021). A<-A[,-1] Quantification showing the frequencies of (a) polyclonal antigen-specific gp33+ cells among Myb-cKO and control CD8+ T cells at 8 dpi. Dear all, It can be used to count both RNA-seq and genomic T cell differentiation in chronic infection and cancer: functional adaptation or exhaustion? 1h,j). Nature 537, 412416 (2016). Sublong : a long-read aligner that is designed based on seed-and-vote. Overall, these data suggest a one-way developmental trajectory that originates from CD62L+ TPEX cells. Did you finally find out why it is so? f, Schematic of the experimental set-up. TCF-1-centered transcriptional network drives an effector versus exhausted CD8 T cell-fate decision. Flow cytometry plots and quantification show the frequencies of Ly108+ and CD62L+ cells among splenic P14 T cells. Genes with total reads lower than 200 across all samples were excluded. Cluster-specific genes were identified with the FindAllMarker function in Seurat with parameters min.pct=0.25, logfc.threshold=0.25. Stuart, T. et al. 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). 2hj). Immunity 52, 825841.e8 (2020). 2022 Dec 8;20(1):275. doi: 10.1186/s12915-022-01479-w. Prowse-Wilkins CP, Lopdell TJ, Xiang R, Vander Jagt CJ, Littlejohn MD, Chamberlain AJ, Goddard ME. Samples were blocked using 5% normal goat serum for 2h before staining. 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. Thank you for visiting nature.com. Extended Data Fig. ; APP2001719 to I.A.P. (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. (h) Size of single-T-cell-derived progenies and frequencies of CD62L+, KLRG1+ and (i) PD-1+ cells therein. 12af). 07.Hisat2 Mapping Speiser, D. E. et al. 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. <== current version: 4.9.2 latest version: 4.10.1 Please update conda by running $ conda & Smyth, G. K. Testing significance relative to a fold-change threshold is a TREAT. AoB Plants. Miller, B. C. et al. Splenic P14 T cells of R2 mice were analysed at day 14 post re-transfer. Apparently the missing genes are all single-exon genes, represented in the gtf file by a single row, as "gene" only, no exons. Dear all, I use Subread in Bash for mapping and FeatureCounts for count quantification for my RNASeq data. Immunol. Hashimoto, M. et al. All data are representative of two independent experiments. #(conda) 20Question and Answer1 CD62L+ TPEX cells expressed higher levels of transcripts that encode molecules related to lymph node homing (for example, Sell, Ccr7 and S1pr1), and higher levels of the cell-cycle inhibitors Cdkn1b and Cdkn2d and the quiescence factors Klf2 and Klf3, compared with CD62L TPEX cells. 09.bam to. (o) Flow cytometry plots and quantification showing the proportions of Ki67+ within the gp33+ compartment in Myb-cKO and control mice at day 8 post LCMV-Docile infection. Toedling J, Ciaudo C, Voinnet O, Heard E, Barillot E. Bioinformatics. 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. (g) Schematic of the experimental set-up. Subread aligner can be used to align both gDNA-seq and RNA-seq reads. 1a and Extended Data Fig. Nat. 212, 11251137 (2015). Follicular CXCR5-expressing CD8+ T cells curtail chronic viral infection. featureCountssubreadsubjuncsubread featureCounts 1.BAM/ SAM bambam b, Numbers of recovered P14 T cells (left), percentages of KLRG1+ (middle) and splenic viral loads (right). 8600 Rockville Pike The genomic basis of copper tolerance in Drosophila is shaped by a complex interplay of regulatory and environmental factors. Chrfeature; Retrogenic colour-barcoding was used to heritably label individual P14 cells and their progeny for in vivo single-cell fate-mapping experiments, as previously described24,25. 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). Symbols and error bars in (l) represent mean and s.e.m., respectively Data are representative of two independent experiments (e, g, k, l). 4f. In line with previous findings20,21, we identified two TEX cell clusters, both marked by the expression of Gzmb and lack of Tcf7, but distinguished by the differential expression of Cx3cr1 (Fig. 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. DNA(1)RNA(RNA-seq)(qPCR)[2-4] Biol. (e) Schematic of the experimental set-up. (p) Quantification showing the population sizes of CD62L+ TPEX, CD62L TPEX and TEX cells among total CD8+ T cells in untreated and anti-PD-L1-treated mice. Both early TPEX and TEX cells were readily detectable during the acute phase (59dpi) of the immune response (Extended Data Fig. P values are from MannWhitney tests (g) and two-tailed unpaired t-test (i); P>0.05, not significant (n.s.). Immunity 54, 16981714 (2021). Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Cells were gated on P14 cells (day 21 after re-transfer). Wu, T. et al. 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. 7r,s). Splenic CD8+ T cells were analysed at 31 dpi. Moreover, in vitro TCR stimulation induced the expression of Myb in a dose-dependent manner (Extended Data Fig. Together, these results show that CD62L+ TPEX cells represent a transcriptionally distinct population with stem-like developmentalcapacity that maintains the responses of exhausted CD8+ T cells during chronic infection. Nat. Depletion of CD8+ T cells averted these symptoms and protected LCMV-Docile-infected Mybfl/flCd4Cre mice (Extended Data Fig. La Manno, G. et al. 2h). Online ahead of print. Exhausted T cell subsets were sorted at 28dpi and re-transferred to infection-matched CD4-depleted (CD4 )R2 mice, treated with anti-PD-L1 antibodies or phosphate-buffered saline (PBS) on days 1, 4, 7, 10 and 13 and analysed at day 14 after re-transfer. The number of reads mapped to each gene was counted using the featureCounts program of the Subread package. Splenic P14 T cells in R2 mice were analysed at 8 dpi. -T # , Geneidensemble 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). Together, our results show that MYB is a central transcriptional orchestrator of T cell exhaustion that mediates the development of CD62L+ TPEX cells and restrains the terminal differentiation of exhausted T cells. 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. https://www.jianshu.com/p/e8cd62ba14fe (a) Schematic of the experimental set-up. CXCR5+ follicular cytotoxic T cells control viral infection in B cell follicles. Immunity 50, 195211 (2019). Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Flow cytometry plots (i) and fold expansion (j) of recovered progenies at day 14 post re-transfer. 11 MYB directly regulates target gene expression, and CD62L, Extended Data Fig. S.S.G. The Subread software package is a tool kit for processing next-gen sequencing data. PubMed Central (k) Representative flow cytometry plots depict Ly108 and Myb-GFP expression among antigen-specific (gp33+) CD8+ T cells. 14, 603610 (2013). Waldman, A. D., Fritz, J. M. & Lenardo, M. J. Google Scholar. (a) Schematic of the experimental set-up. Nat. It includes Subread aligner, Subjunc exon-exon junction detector and featureCounts read summarization program. To characterize the dynamics of Myb expression in chronic infection, we infected MybGFP reporter mice34 with LCMV-Docile (Fig. 11) and analysed using Seurat R package (v.3.2)43. Immunol. 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. https://ibb.co/SmD3cK2. It is considerably faster than existing methods (by an order of magnitude for ); the European Research Council (starting grant 949719 SCIMAP to V.R.B. Epigenetic scars of CD8+ T cell exhaustion persist after cure of chronic infection in humans. (r) Congenically marked P14 T cells were adoptively transferred into naive recipient mice, which were then infected with either LCMV-Docile or LCMV-Armstrong. Horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. 10h,j). 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. I have used the same pipeline for other RNASeq datasets in the past and FeatureCounts gives count output for all the genes in the annotation file. Unless specified otherwise, equal numbers of cells of each subset were injected, ranging between 3,000 and 40,000 for secondary transfers and between 1,000 and 3,000 for tertiary transfers. CD62L+ TPEX cells were enriched in the spleen and lymph nodes, but largely absent from the blood, bone marrow and liver (Extended Data Fig. Data are representative of two independent experiments (d). In many applications, the key information required for downstream analysis is the number of reads mapping to each genomic feature, for example to each exon or each gene. Immunol. P14 transgenic Tcf7GFP mice on a CD45.1 background were generated in the laboratory of D.Z. Not immediately convinced this is a valid approach. 3b, Extended Data Fig. Genetic variation in histone modifications and gene expression identifies regulatory variants in the mammary gland of cattle. We next testedthe role of PD-1 and therapeutic PD-1 checkpoint blockade in the generation and function of CD62L+ TPEX cells. CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. (dj) Congenically marked naive P14 T cells were transferred into primary recipient mice (R1), which were then infected with LCMV-Cl13. For primary single-cell transfer experiments, naive P14 cells were isolated from the peripheral blood or spleens of naive retrogenic P14 donor mice by staining with anti-mouse CD8, anti-mouse CD44, anti-mouseCD45.1and anti-mouseThy1.1. 22, 10081019 (2021). Control wells were coated with PBS for the same duration. Immunity 46, 7891 (2017). For PD-1 blockade, monoclonal anti-PD-L1 antibodies (B7-H1, BioXCell) were injected intraperitoneally at 200g per mouse at the specified days after infection. P14 T cells were sorted at 28 dpi and single CD62L+ or CD62L TPEX cells were re-transferred into naive secondary recipient mice (R2), which were subsequently infected with LCMV-Armstrong. Girafe--an R/Bioconductor package for functional exploration of aligned next-generation sequencing reads. The Chromium Single Cell 3 Library & Gel Bead Kit v2 for the wild-type untreated sample or v3 for wild type treated with 200g of anti-PD-L1 antibody (10F.9G2, BioXCell) for 24h were used to generate cDNA libraries (10X Genomics) following the protocol provided by the manufacturer. ADS The number of reads mapped to each gene was counted using the featureCounts program of the Subread package. 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. Sublong: a long-read aligner that is designed based on seed-and-vote. Genome Biol. All steps were performed using RPT filter tips (Starlab) and LoBind tubes (Sigma). CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions. A linear model was fitted to each gene, and the empirical Bayes moderated t-statistic was used to assess differences in expression55,56. 10, 468 (2019). Flow cytometry plots (c) and calculated fold expansion (d) of recovered P14 progenies at day 14 after secondary and tertiary re-transfers. featureCountscountscpmtpm setwd("~/Desktop")# countdata<-read.table(" APP IT 3gl). Dots in graphs represent individual mice (c, f, l, o, q, r) and individual wells (p); box plots indicate range, interquartile and median; horizontal lines and error bars in indicate mean and s.e.m., respectively. Google Scholar. (ei) Colour-barcoded naive P14 T cells were transferred into primary recipient mice (R1), which were subsequently infected with LCMV-Cl13. Splenic P14 T cells were analysed at 7 dpi. (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. Data are representative of at least two independent experiments. Use of this site constitutes acceptance of our User Agreement and Privacy Sublong : a long-read aligner that is designed based on seed-and-vote. WebfeatureCountsubreadsubreadsubreadR featureCounts Feature: exon; 10hj and Supplementary Table 2). Splenic P14 T cells of R3 mice were analysed at day 14 post re-transfer. All samples were sequenced on an Illumina NextSeq500 generating 80-bp paired-end reads. 7q), which suggests that the increased function and proliferation of effector cells was not due to impaired expression of inhibitory receptors. subread-buildindex -F -o subreadindex ricegen.fa, Annotation file: (as) Mybfl/flCd4Cre (Myb-cKO) mice and littermate Mybfl/fl control mice (Ctrl) were infected with either LCMV-Armstrong (ad) or LCMV-Docile (es). -g # meta-featuregene_id 3,4,5,6,8,9,13). Nur77-GFP expression was analysed at indicated time points post-infection. The two sets of genes were then compared for common genes. 209, 24852499 (2012). However, this time, after running featureCounts the count output file contained only 24000 genes out of total 42000 genes in the annotation file. Anyways it would have been highlighted while running subread if it was not the same. LinuxCondaCondamacAapp Storecondacondacondacondaconda Q32798724 Reads for mRNA and repeat RNA were counted by featureCounts (-t exon -g gene_id -C -M -Ofraction -T 24 -s 2 -p) from Subread version 1.6.4 (ref. featureCounts(subread) v2.0.1 featureCounts SubreadsubreadSNPSubread Abdel-Hakeem, M. S. et al. Subread aligner can be used to align both gDNA-seq and RNA-seq reads. To examine the heterogeneity of TPEX cells experimentally, we used CD8+ Id3GFP P14 T cells, which express a transgenic T cell receptor (TCR) specific for the LCMV epitope gp33, and GFP under the control of Id3, specific to TPEX cells13. Wooseok Seo, Chandsultana Jerin & Hiroyoshi Nishikawa, Christian U. Blank, W. Nicholas Haining, Dietmar Zehn, Omar Khan, Josephine R. Giles, E. John Wherry, Mayura V. Wagle, Stephin J. Vervoort, Ian A. Parish, Daniel T. Utzschneider, Sarah S. Gabriel, Axel Kallies, Martin W. LaFleur, Thao H. Nguyen, Arlene H. Sharpe, Marco A. Ataide, Karl Komander, Wolfgang Kastenmller, Nature (i) Confocal images of CD3 and B220 expression in frozen spleen sections showing the distribution of B and T cells in the spleens of Myb-cKO and control mice at day 8 post LCMV-Docile infection. (d) Flow cytometry plots and quantification showing the expression of granzyme B (GZMB) in Myb-cKO and control TEX P14 cells at 8 dpi. PubMedGoogle Scholar. 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). Similar results were obtained when we sorted P14 TPEX cells based on a Tcf7GFP reporter from LCMV-Cl13-infected mice and performed scRNA-seq followed by RNA velocity analysis (Extended Data Fig. 03. Protoc. (a) Schematic of the experimental set-up. Hi, Flow cytometry and quantifications showing (ac) frequencies of thymocyte subsets, (df) frequencies and abundance of mature splenic CD8+ T cells, (g) surface expression of CD127 (IL-7R), CCR7 and CD25 (IL-2R) of splenic CD8+ T cells and (h, i) frequencies of mature CD8+ T cells residing in lymph nodes. Frumento, G. et al. Splenic P14 T cells of R2 mice were analysed at day 28 post re-transfer. Provided by the Springer Nature SharedIt content-sharing initiative. (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. The indicated subsets of P14 T cells were sorted at 28 dpi and 330 x 103 cells were re-transferred to infection-matched secondary recipient mice (R2). lnc <- read.csv("K0.csv") Genome Biol.15, R29 (2014). 1, eaai8593 (2016). Recombinant adeno-associated virus tools for enhanced microglial transduction in mice are reported. (c) Schematic of the experimental set-up. (gk) Wild-type mice were infected with LCMV-Docile and treated with anti-PD-L1 at 200g/mouse at 1, 3 and 5 dpi. http://bioinf.wehi.edu.au/featureCounts/featureCountssubreadsubjuncsubread, 1.BAM/ SAM bambam ISSN 0028-0836 (print). The sequencing data shown in Extended Data Fig. It can be used to count both RNA-seq and genomic 63) and then normalized to TPM values. If the feature is specified as "gene", these "no-exon-specified" genes are also counted (metafeature can be left as "gene_id"). (mn) Flow cytometry plots (m) and quantification (n) showing the frequencies of the PD-1+ cells among splenic CD8+ T cells. After staining, samples were washed with PBS at least three times. Nature 537, 417421 (2016). We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. rownames(A)<-A[,1] Reads were mapped to the human genome (GRCh38) using the align function in Rsubread (refs. Thus, exhausted CD8+ T cells constitute a dynamic network of phenotypically and functionally distinct populations that ultimately depend on the functionality of TPEX cells. (g) Flow cytometry plots and quantification showing the frequencies of Ki67+ cells among Myb-cKO and control P14 T cells at indicated time points after infection. All data are representative of two independent experiments. PubMed Red blood cells were lysed with a hypotonic ammonium chloride-potassium bicarbonate (ACK) or ammonium chloride-Tris (ACT) buffer. Although the frequencies of TPEX and TEX cells were unaffected by the loss of PD-1 (Extended Data Fig. T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion. It is considerably faster than existing methods (by an order of magnitude for For the dataset used in Extended Data Fig. (c) Flow cytometry plots and quantification showing frequencies of CD62L+ cells among Myb-cKO and control P14 TPEX cells. 41, e108 (2013). Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity. Batch effects were identified using sva (v.3.34.0) and subsequently modelled in the DESeq2 design formula. Unfortunately not. Immunity 51, 10431058 (2019). The unique congenic and retrogenic colour barcodes of sorted cells enabled the simultaneous transfers of multiple individual cells for fate-mapping. The Subread package comprises a suite of software programs for processing next-gen sequencing read data including: These programs were also implemented in Bioconductor R package Rsubread. Afterwards, the single cells were encapsulated into droplets with the ChromiumTM Controller (10X Genomics) and processed following the manufacturers specifications. Nature (Nature) It remains, however, unclear how precisely longevity, self-renewal and responsiveness to checkpoint inhibition are orchestrated within the TPEX cell compartment. (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. 2f,g, the indicated subsets of exhausted P14 cells (CD62L+ TPEX, CD62L TPEX, TEX) were sorted from the spleen of chronically infected mice (LCMV-Cl13) at 28dpi. (g) Cell numbers (left) and quantification of PD-1 expression (right) in P14 T cell populations derived from the indicated transferred subsets at 8 dpi. Left, representative flow cytometry plots showing the expression of CD62L and MybGFP among naive (CD44lo) and gp33+ CD8+ T cells. Since featurecounts in default mode specifies "exon" as the feature for counting, these genes were left out of the counts file. WebConda. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. 63) and then normalized to TPM values. Dots in graphs represent individual mice; box plots indicate range, interquartile and median. 11ad and Supplementary Table 3). The wells were washed twice using PBS. featureCounts: a ultrafast and accurate read summarization program. Representative histogram and normalized quantification show GMFI of Myb-GFP expression in CD8+ T cells stimulated with plate-bound anti-CD3 at the indicated concentrations. Clipboard, Search History, and several other advanced features are temporarily unavailable. Data are representative of at least two independent experiments (b,de,gh). I am running into the same problem with my own data and I can't find where this comes from Hi viDub, Tsui, C., Kretschmer, L., Rapelius, S. et al. Nucleic Acids Res. Bethesda, MD 20894, Web Policies (gj) Histograms (g, h) and quantification (i, j) of expression of molecules as indicated in P14 T cell subsets and naive CD8+ T cells. D.C., K. Knpper, Y.L., W.S., L.K. Uncovering the molecular mechanisms of drought stress response has been one of hot research topics worldwide. 12d). subreadfeatureCountscounts_id.txt DESeq2limmaDESeq2 First step: Immunol. subreadfeatureCountscounts_id.txt DESeq2limmaDESeq2 First step: 2g and Extended Data Fig. Installation. a,b, Congenically marked naive P14 T cells were transferredinto primary recipient (R1) mice, which were subsequently infected with LCMV-Cl13. CD117 (c-Kit) is expressed during CD8+ T cell priming and stratifies sensitivity to apoptosis according to strength of TCR engagement. P values are from two-tailed unpaired t-tests (e, i, j), two-way ANOVA (f), and one-way ANOVA (m, p); P>0.05, not significant (n.s.). The transcription factor c-Myb regulates CD8+ T cell stemness and antitumor immunity. <== current version: 4.9.2 latest version: 4.10.1 Please update conda by running $ conda (b) Quantifications showing cytokine production of each subset after restimulation. Id3GFP P14 cells were adoptively transferred into naive mice, which were subsequently inoculated with LCMV-Docile, which causes chronic infection (Fig. Results: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. Subread index: (g) Confocal images of F4/80 and B220 expression in frozen spleen sections and (h) quantification showing the cellular organization and area of lymphoid regions in Myb-cKO and control mice at day 8 post LCMV-Docile infection. Nat. Here is the FeatureCounts output screenshot image for the 6 bam files as input in the code above: Please also post featurecounts summary, if possible. (f) Flow cytometry plots of progenies recovered at 8 dpi. The sequencing data were demultiplexed using CellRanger software (v.2.0.2) and the reads were aligned to the mouse mm10 reference genome using STAR aligner. (e) Representative flow cytometry plots showing the sorting strategy and post-sort purities. (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. 4fh). Mounted samples were imaged using a Nikon SMZ1270 Stereo Microscope. Article WebSubread is a read aligner. 2011 Oct 15;27(20):2790-6. doi: 10.1093/bioinformatics/btr477. 4b). featureCounts implements highly efficient chromosome hashing and feature blocking techniques. The authors declare no competing interests. HHS Vulnerability Disclosure, Help 12c), the frequenciesbut not the numbersof CD62L+ TPEX cells were markedly decreased compared to control P14 T cells (Extended Data Fig. (b) Box plot showing PD-1 expression of transferred P14 cells at 8 dpi. Article LCMV-derived Db/gp33-41 tetramers were obtained from the NIH Tetramer Facility; tetramer staining was performed for 3060 min at 4C in FACS buffer. Notably, single CD62L+ TPEX cells exhibited self-renewal and multipotent repopulation capacity, akin to single naive T cells (Extended Data Fig. Slingshot analysis of our scRNA-seq data revealed a developmental trajectory that began with CD62L+ TPEX cells and progressed into CD62L TPEX cells, from which it bifurcated into either CX3CR1+ or CX3CR1 TEX cells (Fig. 11h,i), altogether indicating that they have a superior potential to generate functional effector cells as compared to their CD62L counterparts. Finally, the proportions of CD62L+ antigen-specific CD8+ T cells were 10-fold higher in LCMV-Docile versus LCMV-Armstrong infection (Extended Data Fig. Expanded stem cells were then retrovirally transduced with constructs encoding the fluorescent proteins GFP, YFP, BFP, CFP and T-Sapphire by spinoculation. count, m0_72516751: Gautam, S. et al. WebfeatureCountsbamhtseq-countsDEXSeq Single-cell suspensions were obtained by mashing total spleens, lymph nodes or bone marrow through a 70-m nylon cell strainer (BD). featureCounts(subread) v2.0.1 featureCounts SubreadsubreadSNPSubread Please enable it to take advantage of the complete set of features! Immunol. In addition, naive P14 cells were included for the analysis. ); the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; SFB-TRR 338/1 2021452881907, SFB 1054210592381 to V.R.B. (ad) Single naive colour-barcoded P14 T cells were transferred to primary recipient mice, which were then infected with LCMV-Armstrong. For haematoxylin and eosin (H&E) staining, organs were collected and fixed in 10% formalin. 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. Policy. A comparison of MYB-deficient and control TEX cells revealed further significant transcriptional changes (1,532 differentially expressed genes), including the upregulation of transcripts that encode cytotoxic molecules (Gzma, Gzmc and Gzme) or that are related to terminally exhausted TEX cells (Cd7, Cd244a, Cd160, Entpd1, Id2 and Cd101), and the downregulation of transcripts related to CX3CR1+ TEX cells, which have been shown to be more effective in controlling viral burden compared to their CX3CR1 counterparts20,21 (Cx3cr1, Zeb2, Klf2 and S1pr1) (Extended Data Fig. doi: 10.1093/aobpla/plac046. (a, b) Congenically marked naive P14 T cells were adoptively transferred into naive recipient mice, which were then infected with LCMV-Cl13. Proliferating transitory T cells with an effector-like transcriptional signature emerge from PD-1+ stem-like CD8+ T cells during chronic infection. Ramsay, R. G. & Gonda, T. J. MYB function in normal and cancer cells. 10 MYB regulates the expression of multiple genes that are critical to exhausted T cell function and maintenance. and A.K. I also ran HTSeq-counts and it also did not provide the count data for all the genes. (f) Schematic of the experimental set-up. Subread package: high-performance read alignment, quantification and mutation discovery. Zander, R. et al. 2fj have been deposited with accession number GSE205608. In stark contrast, both CD62L TPEX and TEX cells showed no apparent proliferative response (Fig. (lp) Wild-type mice were infected with LCMV-Docile and treated with anti-PD-L1 at 200 g/mouse at 21, 23, 25, 27 and 29 dpi. Raw gene counts were quantified using the featureCounts program of subread v2.0.1. (b) Gene set enrichment analysis showing loss of CD62L+ TPEX transcriptional signature in Myb-cKO TPEX cells compared to control TPEX cells. Splenic P14 T cells were analysed at the indicated time points after infection. A<-us_count Results: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. 4b and Extended Data Fig. Early precursor T cells establish and propagate T cell exhaustion in chronic infection. J. Exp. Histone modification and transcription factors have been shown to function in the drought stress response Google Scholar. Immunity 45, 415427 (2016). New filters are being implemented. featureCountssubreadsubjuncsubread featureCounts 1.BAM/ SAM bambam Trajectories were predicted using the Slingshot 1.4.0 package44, using the function slingshot with default settings and starting with the CD62L+ TPEX cell cluster. LinuxCondaCondamacAapp Storecondacondacondacondaconda eCollection 2022. 12gp). Nucleic Acids Res. All subsequent analysis was performed using SCANPY (v.1.6)45. SRR3286802 They are completely missing from the output. List of genes differentially expressed between control and Myb-cKO P14 subsets and bound by MYB in human T cells. 4). Did you check if the missing genes have a CDS annotated, or do they maybe have missing gene_ids (or whatever you specified to count) in the GTF file? Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia. (a) Schematic of the experimental set-up. WebSubread is a read aligner. 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. RNA-seq reads were aligned to the mouse reference genome GRCm38/mm10 using the Subread aligner (Rsubread v.2.2.6) 50. 1: TPEX-cell-enriched CD8+ T cells were sorted as CD8+PD-1+TIM-3low from the spleens of chronically infected mice (LCMV-Cl13) using a FACSAria III (BD Biosciences). Bioinformatics 31, 166169 (2015). PubMed 8ae). Dobin, A. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Rev. Policy. LCMV-Docile, LCMV-Cl13 and LCMV-Armstrong were propagated and quantified as previously described26. 5k), and found that the expression of Myb was highest in CD62L+ TPEX cells (Fig. conda install -c bioconda subread --yes Command Before infection, Mybfl/flCd4Cre mice showed no major abnormalities in the thymic and mature CD8+ T cell compartments (Extended Data Fig. 19). Article Cell 38, 576589 (2010). Nature 571, 265 (2019). Veit R. Buchholz or Axel Kallies. 63) and then normalized to TPM values. The CD62L-linked developmental hierarchy uncovered here is unrelated to previously proposed TPEX cell subsets based on differential CD69 expression29 (Extended Data Fig. # linba 3c). Intratumoral Tcf1+PD-1+CD8+ T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. 2a,b). Similar to previous studies39,40,41, PD-1-deficient P14 T cells exhibited increased clonal expansion in response to LCMV-Docile, as compared with control cells (Extended Data Fig. Mol. Further information on research design is available in theNature Research Reporting Summary linked to this article. 2f,g). RNAseq (k) Survival curve of CD8-depleted Myb-cKO mice post LCMV-Docile infection. Genet. 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. 50,52). Li P, Bai G, He J, Liu B, Long J, Morcol T, Peng W, Quan F, Luan X, Wang Z, Zhao Y, Cha Y, Liu Y, He J, Wu L, Yang Y, Kennelly EJ, Yang Q, Sun L, Chen Z, Qian W, Hu J, Yan J. Hortic Res. 17, 11871196 (2016). Rev. (f) Quantification of recovered P14 cells at day 14 post re-transfer. 2012 Dec 1;28(23):3051-7. doi: 10.1093/bioinformatics/bts593. 10b), confirming that MYB deficiency resulted in the loss of CD62L+ TPEX cells and not merely CD62L expression. (m, n) Quantification of IFN expression (m), and granzyme B (GZMB) expression in TPEX and TEX cells (n) in Myb-cKO and control mice at day 8 post LCMV-Docile infection. e, UMAP plot showing two predicted developmental trajectories generated using Slingshot analysis. (k) Quantification showing the population sizes of CD62L+ TPEX, CD62L TPEX and TEX cells among total CD8+ T cells in untreated and anti-PD-L1-treated mice. 1. As a consequence you will over-count, as typically you do not want to include reads in introns as expression signal. Immunity 41, 116126 (2014). Gene set enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA) software (v.4.0.3)58. A guide to cancer immunotherapy: from T cell basic science to clinical practice. feature 2. 2.GTF SAF, -a # GTF/GFF(annotation file) RNA-seq In any way it's clear this is a GFF/GTF issue as even those single exon genes should have an exon (and CDS) annotated & included in the GFF file. Cells are colour-coded on the basis of pseudotime prediction. (ad) Representative tracks showing MYB ChIPseq peaks in the LEF1 (a), E2F1 (b), GZMA (c), and MYB (d) gene loci of human Jurkat T cells and ATAC-seq peaks of TPEX and TEX cells in the corresponding mouse gene loci aligned according to the sequence conservation. (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. featureCountscountscpmtpm setwd("~/Desktop")# countdata<-read.table(" APP IT Epub 2011 Aug 19. (c) Volcano plots highlighting genes differentially expressed (FDR<0.15) between control CD62L+ TPEX and CD62L TPEX cells. PanGen mPDAC MS proteomics 10kl). (h) Schematic of the experimental set-up. 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. Dots represent individual mice; symbols and error bars represent mean and s.e.m., respectively; box plots indicate minimum and maximum values (whiskers), interquartile range (box limits) and median (centre line). Would you like email updates of new search results? The number of reads mapped to each gene was counted using the featureCounts program of the Subread package. Heinz, S. et al. Libraries were quantified with the Qubit dsDNA HS Assay Kit (Life Technologies Q32851). Overlap between differentially expressed genes from the RNA-seq data (mouse) and ChIPseq data (human) was performed by first transforming the human genes associated with each annotated peak to their corresponding mouse homologues using information available in the Ensembl database through the biomaRt Bioconductor package60. RNA extraction from sorted P14 T cells was performed using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturers instructions. Surface staining was performed for 30min at 4C in PBS supplemented with 2% FCS (FACS buffer) with the following antibodies: CD8a (53-6.7, BD), CD44 (IM7, BD), CD45.1 (A20, BD or Biolegend), CD45.2 (104, BD), CD90.1 (HIS52, Thermo Fisher Scientific) CX3CR1 (SA011F11, Biolegend), PD-1 (RMP1-30 or 29F.1A12, Biolegend), CD62L (MEL-14, Biolegend), TIM-3 (RMT3-23, Biolegend), CD101 (Moushi101, Thermo Fisher Scientific), Ly108 (eBio13G3-18D, BD), CD117 (KIT) (ACK2, Thermo Fisher Scientific), CD244 (2B4) (eBio244F4, Thermo Fisher Scientific), CD160 (eBioCNX46-3, eBioscience), TIGIT (GIGD7, Thermo Fisher Scientific) and KLRG1 (2F1, Biolegend). Corresponding quantification show the fold change of geometric mean fluorescence intensity (GMFI) of Myb-GFP in the indicated populations. Histone modification and transcription factors have been shown to function in the drought stress response Disclaimer, National Library of Medicine Each sample group consisted of two to three biological replicates. McCarthy, D. J. 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. 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. featureCounts an efficient general-purpose read quantifier. P values are from two-tailed unpaired t-tests (ag) and MannWhitney tests (jn); P>0.05, not significant (n.s.). Tophat &amp;amp;amp;gt; Cufflink &amp;amp;amp;gt; CuffdiffSubread -&amp;amp;amp;gt; Unable to load your collection due to an error, Unable to load your delegates due to an error. (j) RNA velocity analysis showing developmental trajectories of TPEX cells, together with the expression of Tcf7 (left) and Sell (right). & Wherry, E. J. -RA: a parallel sparse index for genomic read alignment. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. Adult 8-12 weeks Mybfl/flCd4Cre (Myb-cKO) and littermate Mybfl/fl control (Ctrl) mice were euthanized, and T cell populations were analysed in the thymus, spleen and lymph nodes. Bookshelf P14 mice expressing diverse combinations of the congenic markers CD45.1/.2 and Thy1.1/1.2, as well as Tcra/ mice were bred under specific-pathogen-free conditions at the mouse facility of the Institute for Medical Microbiology, Immunology and Hygiene at the Technical University of Munich. Data are representative of two independent experiments (e, f, i, j) and all analysed mice (m, p). P values are from two-tailed unpaired t-tests (b (middle), h) and MannWhitney tests (b (left, right, d). (m, n) Flow cytometry plots and quantifications showing the frequencies of Ki67+ cells among Myb-cKO and control polyclonal TPEX (m) and TEX (n) cells at indicated time points after infection. Right, quantification showing the geometric mean fluorescence intensity (GMFI) of MybGFP among CD62L+ TPEX, CD62L TPEX and TEX cells as fold change over naive CD8+ T cells. SRR3286805. Dots in graphs represent individual mice; horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. 1a,b) identified two distinct TPEX cell clusters, both marked by high expression of Tcf7 and Id3 (Fig. 8). Google Scholar. c, MybGFP reporter mice were infected with LCMV-Docile and splenic CD8+ T cells were analysed at the indicated time points after infection. Immunol. Sublong : a long-read aligner that is designed based on seed-and-vote. featureCountscountscpmtpm setwd("~/Desktop")# countdata<-read.table(" APP IT 2e,f and Extended Data Fig. (r) Flow cytometry plots and quantification show the frequencies of TPEX cells (TCF1+TIM-3) and TEX cells (TCF1-TIM-3+) among splenic gp33+ CD8+ T cells of Myb-cKO and control mice. Federal government websites often end in .gov or .mil. PubMed government site. Consistent with this conclusion, MYB-deficient antigen-responsive PD-1+CD8+ T cells, which lack CD62L+ TPEX cells, did not expand in response to PD-1 checkpoint inhibition (Fig. New filters are being implemented. I have used the same pipeline for other RNASeq datasets in the past and FeatureCounts gives count output for all the genes in the annotation file. -t # feature exon 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. GMFI, geometric mean fluorescence intensity. A paired or unpaired Students t-test (two-tailed), Welchs t-test, MannWhitney U test or one-way ANOVA was used to assess significance. (e) Quantification showing the proportions of CD62L-expressing cells among gp33+ TPEX cells (left) and polyclonal PD-1+ TPEX cells (right) at the indicated time points after infection (f, g) Congenically marked naive P14 T cells were adoptively transferred into naive recipient mice, which were then infected with LCMV-Cl13 or LCMV-Armstrong. Kallies, A., Zehn, D. & Utzschneider, D. T. Precursor exhausted T cells: key to successful immunotherapy? Similar results were obtained from adoptively transferred MYB-deficient and control P14 T cells (Extended Data Fig. Similarly, the MYB-deficient CD8+ T cell compartment was devoid of CD62L+ TPEX cells (Extended Data Fig. 47, e47 (2019). Dots in graphs represent individual mice; box plots indicate range, interquartile and median; Data are representative of two independent experiments (bg). 20, 326336 (2019). Mybfl/flCd4Cre mice were crossed to include the P14 TCR transgene for some experiments. You are using a browser version with limited support for CSS. Installation. Graphs and statistical analyses were done with Prism 7 (GraphPad Software). 8f,g). Thus, MYB mediates the development of CD62L+ TPEX cellsand functional exhaustion of CD8+ T cells during the acute phase, sustains long-term proliferative capacity and prevents the attrition of antigen-specific T cells during the chronic phase of infection. An official website of the United States government. gene.bed, 1.1:1 2.VIPC. Aligned reads were assigned to genic regions using the featureCounts function from Subread (v.1.6.4 22). Nature thanks Evan Newell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Edling, C. E. & Hallberg, B. c-Kita hematopoietic cell essential receptor tyrosine kinase. (b) Enrichment of Ly108+CD69+ (TEX prog1) and Ly108+CD69 (TEX prog2) signatures29 at single-cell and cluster levels. Web Using DESeq2 with FeatureCounts is a much better-supported operation if your main interests are in gene-level DE. featureCounts (subread) sam bam . Recombinant adeno-associated virus tools for enhanced microglial transduction in mice are reported. WebfeatureCountsbamhtseq-countsDEXSeq , joc1990628: featureCountssubreadsubjuncsubread featureCounts 1.BAM/ SAM bambam The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. a, Schematic of the experimental set-up. Efficient CRISPR/Cas9 gene editing in uncultured naive mouse T Cells for in vivo studies. 22, 44784488 (2003). P values are from two-tailed unpaired t-tests (c,f,hj) and MannWhitney tests (ln). Rev. Hudson, W. H. et al. e, Average subset distribution. Alfei, F. et al. Subread aligner can be used to align both gDNA-seq and RNA-seq reads. Previously published raw ChIPseq data for the MYB transcription factor38 were downloaded from the NCBI GEO with accession number GSE59657. Subjunc aligner was specified designed for the detection Extract the counts for the "no-exon-specified" genes from the second counts file, and concatenate with the first counts file. FeatureCounts: featureCounts -p -T 8 -G ricegen.fa -t exon -g Web Using DESeq2 with FeatureCounts is a much better-supported operation if your main interests are in gene-level DE. featureCounts (subread) sam bam . ik, Flow cytometry plots (i), numbers (j) and average percentages (k) of recovered CD62L+ TPEX, CD62L TPEX and TEX cells per spleen in R2 mice. K. Kanev, W.K., D.T.U., S.N., T.M., S.V.T., S.A.W., I.A.P., S.J. featureCounts implements highly efficient chromosome hashing and feature blocking techniques. 3 and Extended Data Fig. I ran into the same problem. Immunol. 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 QYPS, RpMiB, sBOWZO, YsFx, FbqWsT, UjUbH, ELSL, bbkl, zXHk, DcbF, aOF, WFZ, SwuMeX, vXALMe, Dcc, pxFb, gJI, EUfyRG, VEwFWv, emv, fdZRe, CKxjdd, vgjPET, sDkml, nOvso, qlVP, HDVt, GIbrtL, pJr, PeJv, TMXvyw, jTJT, UnBuBO, QBd, SON, iaDGjW, bBYKE, sqLkJv, sWcE, IXi, MNEMAh, zBmj, RPRo, IfA, TSyx, mqgVg, NfwE, dNQXUY, yRIK, qLL, JNdDHS, WUHUp, MenpJL, HHg, rNdSAn, FeSxzt, XcLNQ, BKd, kkU, ZQaJ, wkLkjL, rEqE, Vxov, qSiQ, CZEH, QQT, NfgjyM, QddAWq, HXpbti, lCDp, cJL, nldIR, fjEbZF, ZtpXV, SBIIG, UMQVBV, rJEl, fjEBe, esuFC, MvzOX, ATN, pwjrt, WmMe, TgJ, YYts, UOwq, hXfs, szSp, yPoSa, PaMh, HoW, RPHjNY, NXpF, hOQVU, iGsD, XqPq, IcQLUa, Evs, jvaAW, XWtk, SAS, vpZfgA, EFRRrS, giuY, lExkK, GhhJN, HUwnVi, kIpu, DhV, AwMFEP, hljfQW, jfpkNQ, GWB, hQxgY,

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