gene set enrichment analysis algorithm  (Immunome Inc)

Journal: Nature Communications

Article Title: Transcriptome and chromatin landscape of iNKT cells are shaped by subset differentiation and antigen exposure

doi: 10.1038/s41467-021-21574-w

Figure Lengend Snippet: a chromVAR computed deviation in ATAC-seq signal (Z-score) at regions containing indicated transcription factor motifs between the indicated cell populations from spleen and lung. Motifs with a p value less than 1e-25 are shown and families and representative members are labeled. Tissue source of the samples indicated at the bottom of the diagram; columns indicate number of replicates. b PCA of RNA-seq data comparing lymphoid cell types isolated from lung and spleen. c Heat map depicting the lung gene signature from Supplementary Fig. with normalized transcript levels from RNA-seq data from the indicated cell types from lung and spleen. d RNAseq read tracks in the Bhlhe40 , Tnfaip3 , and Fosl2 loci in samples from lung and spleen from indicated cell types. e GSEA of pre-ranked comparisons of genes differentially expressed in lung iNKT cell subsets comparing each cell type within the lung to its counterpart in the spleen.

Article Snippet: Principal Component Analysis (PCA) was performed using the ‘prcomp’ function in R. Data were also analyzed using the Pre-ranked Gene Set Enrichment Analysis algorithm (Broad Institute and University of California), as well as the Consensus Path Database platform (Max Planck Institute).

Techniques: Labeling, RNA Sequencing, Isolation

Journal: Nature Communications

Article Title: Transcriptome and chromatin landscape of iNKT cells are shaped by subset differentiation and antigen exposure

doi: 10.1038/s41467-021-21574-w

Figure Lengend Snippet: iNKT cells from αGalCer-injected mice either selected for PD-1 and CXCR5 expression indicating similarity to follicular helper (FH) T cells (NKT FH ) or negative for both markers and similar to effectors (eff) (NKT eff ) were sorted from the spleen of mice 6 days after injection (i.v.) with αGalCer. a Scatterplots of mean ATAC-seq counts per peak comparing antigen-experienced NKT FH and NKT eff (top left) or pairwise comparisons of differentially accessible regions of chromatin for each of the sorted populations from antigen exposed mice compared to the corresponding subsets from unimmunized mice. Data from unimmunized mice are the same as depicted in Fig. . Colors indicate differentially accessible regions defined by limma/voom (details in Methods). b ATAC-seq coverage (range of 0–600 for all samples) comparing the Il21 and Pdcd1 loci from unimmunized splenic iNKT cell subsets, splenic NK cells, and NKT FH and NKT eff from αGalCer-treated mice. NFAT ChIP-seq analysis of CD8 + splenic T cells with and without PMA/ionomycin stimulation included for comparison . c Left, k -means clustering of relative ATAC-seq density (counts per million mapped reads/kb, log fold change from the mean) identifies ten groups of accessible regions that vary similarly (rows), two sets for splenic NKT1, two for splenic NKT2, two for splenic NKT17, six for NKT FH , and three for NKT eff . Columns indicate the number of replicates. Right, motifs enriched in clusters of accessible regions. All motifs with a HOMER log p value less than –15 and found in 10% or more regions in at least one cluster are shown. d PCA analysis of RNA-seq data comparing NKT FH to NKT eff from αGalCer-immunized mice, as well as to total iNKT cells from unimmunized mice. e PCA analyses of RNA-seq data comparing splenic NKT1, NKT2, or NKT17 samples to spleen NKT FH cells. f Top: Plot of the distribution of genes upregulated in mainstream GC T FH vs T H 1 in a list of genes ranked by relative expression (directional p value) in NKT FH vs splenic NKT1 cells using GSEA. Bottom (left and right): Plots of genes differentially regulated between CD8 + effector vs memory against a directional p-ranked file comparing αGalCer-stimulated NKT FH vs NKT eff . Normalized Enrichment Scores (NES) and q values were determined by the pre-ranked GSEA algorithm. g Expression of reporter in T-bet fate-mapping mice by NKT FH and NKT eff cells 6 days post antigen exposure, and NKT1 and NKT2 cells and total iNKT cells from unstimulated mice; n = 6 mice per group, error bars depict SEM. Quantification on right, statistical significance ( p < 0.0001) assessed via Kruskal–Wallis test.

Article Snippet: Principal Component Analysis (PCA) was performed using the ‘prcomp’ function in R. Data were also analyzed using the Pre-ranked Gene Set Enrichment Analysis algorithm (Broad Institute and University of California), as well as the Consensus Path Database platform (Max Planck Institute).

Techniques: Injection, Expressing, ChIP-sequencing, Comparison, RNA Sequencing