Journal: Nature

Article Title: The type 2 cytokine Fc–IL-4 revitalizes exhausted CD8 + T cells against cancer

doi: 10.1038/s41586-024-07962-4

Figure Lengend Snippet: a , b , Real-time ECAR analysis ( a ) and average basal glycolysis, glycolytic capacity and reserve ( b ) of ex vivo-induced CD8 + T TE cells ( n = 5 biological replicates). mpH, milli-pH. c , Volcano plot of altered metabolites in ex vivo-induced CD8 + T TE cells treated with Fc–IL-4 ( n = 4 biological replicates) versus PBS ( n = 3 biological replicates). d – f , Experimental setting as described in Fig. . Shown are unsupervised UMAP clustering of PMEL CD8 + TILs based on the 1,667 genes involved in KEGG-defining metabolic pathways ( d ), cell proportion in each cluster ( e ) and systematic expression comparison of carbohydrate metabolisms among top four clusters ( f ). g , h , T cell counts ( g ) and frequencies of granzyme B + IFNγ + ( h ) among ex vivo-induced CD8 + T TE cells with or without 2-DG ( n = 4 biological replicates). i , j , Schematic illustration of single-cell ATAC and gene coprofiling of IL-4 versus PBS-treated ex vivo-induced CD8 + T TE cells and a joint ATAC–gene UMAP ( i ), and volcano plot showing differentially active motifs ( j ). k , l , Experimental setting as described in Fig. . Shown are signalling pathways regulated by DEGs ( k ) and top 20 ranked upstream regulators predicted from DEGs ( l ) in PMEL CD8 + TILs. m , Western blot analysis of indicated proteins in ex vivo-induced CD8 + T TE cells ( n = 3 biological replicates). n – p , Relative basal glycolysis ( n ) ( n = 5 biological replicates), T cell counts ( o ) and granzyme B MFI ( p ) ( n = 3 biological replicates) in Fc–IL-4-treated ex vivo-induced OT1 and OT1 STAT6-KO CD8 + T TE cells (normalized by that in the PBS group) with or without indicated inhibitors. All data represent mean ± s.e.m. and are analysed by two-sided unpaired Student’s t -test ( b and g , h ), two-tailed Mann–Whitney test ( j ), right-tailed Fisher’s exact test ( k ) or one-way ANOVA and Tukey’s test ( n – p ). Schematics in i created using BioRender ( https://Biorender.com ).

Article Snippet: Single-cell coprofiling of epigenomic landscape and gene expression in the same single nuclei was performed using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics).

Techniques: Ex Vivo, Expressing, Comparison, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: The type 2 cytokine Fc–IL-4 revitalizes exhausted CD8 + T cells against cancer

doi: 10.1038/s41586-024-07962-4

Figure Lengend Snippet: a , Quality assessment of sequenced data from IL-4 or PBS conditions, featuring TSS enrichment score, insert size distribution, unsupervised clustering analysis of ATAC and gene datasets, and corresponding count distribution. Consistent performance is observed with negligible batch effect. b , Gene or ATAC expression UMAP of all the single cells color-coded by their respective conditions. c , d , Expression of functional cytotoxicity ( c ) and survival ( d ) gene markers on the joint UMAP in Fig. , along with comparisons of corresponding accessible peaks between conditions. Statistical analyses were performed using two-sided unpaired Student’s t-test. e , Experimental setting was described in Fig. . Shown is the mechanistic networks associated with the significant activation of selected upstream regulators in Fc–IL-4 treated PMEL CD8 + TILs relative to the PBS condition. z score is computed and used to reflect the predicted activation level (z > 0, activated/upregulated; z < 0, inhibited/downregulated; z ≥ 2 or z ≤ −2 can be considered significant). Statistical analyses are performed using right-tailed Fisher’s Exact Test.

Article Snippet: Single-cell coprofiling of epigenomic landscape and gene expression in the same single nuclei was performed using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics).

Techniques: Expressing, Functional Assay, Activation Assay

Journal: Developmental cell

Article Title: Single-cell epigenome analysis identifies molecular events controlling direct conversion of human fibroblasts to pancreatic ductal-like cells.

doi: 10.1016/j.devcel.2023.08.023

Figure Lengend Snippet: Figure 4. FI-snMultiome method for dissecting the role of defined TFs in reprogramming at single-cell resolution (A) Schematic presentation of the lentiviral expression construct for barcoded TFs and the strategy for capturing the barcodes during 103 Multiome snRNA-seq workflow. UMI, unique molecular identifier. (B) Reprogrammed cells transduced with six barcoded TFs individually or as a pool were harvested at different time points and multiplexed for the analysis of transcriptomic (snRNA-seq) and epigenetic (snATAC-seq) changes from the same cell. Custom TF barcode library was generated by an additional PCR after the pre-amplification step during the 103 Multiome workflow (see STAR Methods), enabling correlation of the TF barcodes with the 103 cell barcodes during downstream analysis. (C) Uniform manifold approximation and projection (UMAP) plots of all cells from different time points (indicated with colors) based on gene expression (left) and chromatin accessibility (middle) separately and their integrated profiles using weighted nearest neighbor (WNN) analysis (right). HFFs transduced with green fluorescent protein (GFP) reporter gene were used as a control. For all FI-snMultiome-seq analyses, 5,399 individual cells from 1 week, 6,921 from 2 weeks, and 10,276 from 48 h and HFFs were used. (D) UMAPs showing the transcript levels of endogenous TFs (left), exogenous TFs detected from their barcodes (middle), and motif accessibility analyzed from the snATAC-seq data (right).

Article Snippet: Since this indexing barcode is close to the poly(A) tail of the transcripts, a large fraction of the barcodes is captured during polyA enrichment and 3’RNA-seq library preparation protocol used in 10x multiome assay kit (10x Genomics).

Techniques: Expressing, Construct, Transduction, Generated, Gene Expression, Control

Journal: Communications Biology

Article Title: One-pot method for preparing DNA, RNA, and protein for multiomics analysis

doi: 10.1038/s42003-024-05993-1

Figure Lengend Snippet: a The ProMTag Multiomics workflow. b TNBC cell lines were put through the ProMTag Multiomics workflow to produce DNA, RNA, and peptides that were analyzed via WGS, RNA-Seq, and mass spectrometry (MS), respectively. For each replicate, we began with a volume of cell lysate that contained 100 µg of protein. c Yields of DNA, RNA, and peptides from each replicate.

Article Snippet: Comparable multiomics sample preparation methods that are commercially available include the Qiagen AllPrep DNA/RNA/Protein kit.

Techniques: RNA Sequencing Assay, Mass Spectrometry

Journal: Communications Biology

Article Title: One-pot method for preparing DNA, RNA, and protein for multiomics analysis

doi: 10.1038/s42003-024-05993-1

Figure Lengend Snippet: The ProMTag Multiomics method was applied to three 5–7 mg pieces of mouse liver ( a – c ), brain ( d – f ), and lung ( g – i ). a , d , g Cleaned-up nucleic acids were run on 1% TAE agarose gels stained with ethidium bromide. b , e , h RINs were determined via a TapeStation RNA assay. c , f , i RNA and DNA yield were determined via Qubit dsDNA and RNA broad range assays, and peptide yield was assayed by a Pierce quantitative fluorometric peptide assay.

Article Snippet: Comparable multiomics sample preparation methods that are commercially available include the Qiagen AllPrep DNA/RNA/Protein kit.

Techniques: Staining