Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: (a) Heatmap of all differentially expression genes (DEGs) using Seurat’s FindAllMarkers function (parameters: logfc.threshold = 0.25, P < 0.05 determined by two-tailed Wilcoxon’s rank sum test) between CD4 T cell subsets in TEA-seq dataset. (b) Heatmap of all differentially accessible peaks using ArchR’s getMarkerFeatures (parameters: FDR< = 0.1, Log2FC ≥ 0.5) between CD4 T cell subsets in TEA-seq dataset. (c) Pseudo-bulk expression values from our confirmatory scRNA-seq dataset from pediatric (Ped), young adult (YA), and older adult naive CD4 T cells of select age-specific genes identified in TEA-seq. (d) Gene expression of SOX4, TOX, CPQ, and STAT4 in bulk-sorted naive CD4 T cells (CD3 + CD4 + CD8 neg CD45RA + CCR7 + CD27 + CD95 neg ) from newborn cord blood (n = 7 donors) and older adult peripheral blood (n = 6 donors) using qRT-PCR. Two-tailed Mann-Whitney test. * P < 0.05, **** P < 0.0001. In panels a–c, values have been scaled (z-score) per gene or peak.

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Expressing, Two Tailed Test, Quantitative RT-PCR, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: a , Heat map of the top 20 DEGs for each age group in individual true naive CD4 + T cells. For visualization, values are scaled ( z score) per gene. Exp, scaled expression. b , Dot plots of average pseudobulk gene expression for select transcripts in true naive CD4 + T cells separated by age ( n = 8 per group; P, pediatric; OA, older adult). The line indicates the median value. P values were determined by a two-tailed Mann–Whitney test. ** P = 0.0006, *** P = 0.0002. c , TF binding motif enrichment comparison between age groups in true naive CD4 + T cells. The P adj of enrichment was determined by hypergeometric testing. ETV1/ETV2, ETS translocation variant 1/2; NFATC3, nuclear factor of activated T cells, cytoplasmic 3; ATF3/ATF7, activating TF 3/7; TCFL5, TF-like 5 protein; CREM, cAMP-responsive element modulator; SPIB, Spi-B TF; SOX4/SOX10, SRY-box TF 4/10. d , ChromVar motif enrichment UMAP plots. Areas enriched for true naive CD4 + T cells in older adults (orange) and children (green) are outlined. dev, deviation. e , Overview of the scRNA-seq confirmatory cohort ( n = 16 per age group). f , RNA-based UMAP plot of naive CD4 + T cells from the confirmatory cohort. g , Average pseudobulk expression of select signature genes in the naive CD4 + T cell subset for each donor across all age groups, including an external cord blood ( n = 3) dataset. Best-fit lines with 95% confidence intervals are shown. AvgExp, average expression.

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Binding Assay, Comparison, Translocation Assay, Variant Assay

Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: a , Identification of subsets within CD8 + CD27 + CD197 + CD45RA + T cells through a trimodal analysis, shown in a 3WNN UMAP plot with the true naive, SCM, MNP-1, MNP-2 and MAIT subsets colored. b , Expression of select RNA and ADT cell type markers, shown in 3WNN UMAP plots. The modality of detection is indicated in square brackets. Density, gene-weighted 2D kernel density. c , Chromatin accessibility tracks of the IFNG gene region in naive CD8 + T cell subsets, showing normalized read coverage. d , Bar plot (median value shown) of the frequencies of naive CD8 + T cell subsets within the overall naive CD8 + compartment by age group ( n = 8 per group). P values were determined by a two-tailed Mann–Whitney test with the Holm–Sidak multiple-comparison method. * P < 0.05 ( P = 0.02), ** P < 0.01 ( P = 0.003), *** P < 0.001 ( P = 0.0008). e , Age-specific composition of the non-naive compartment found within naive CD8 + T cells. f , 3WNN UMAP plot of all T cells overlaid with naive CD8 + T cell subsets and separated by age. Only cells from the naive CD8 + T cell compartment of children (left) or adults (right) are colored; all other cells are gray. g , Comparison of differential chromatin accessibility across all CD8 + T cell subsets (24,874 features). For visualization, all values are scaled ( z score) per differential region. h , Dot plot of select DEGs across naive CD8 + T cell subsets. The size of points corresponds to the fraction of cells expressing each gene; color corresponds to average expression. AvgExp, scaled average expression. i , Identification of the MNP-2 subset through gene expression profiling in the scRNA-seq confirmatory cohort. Density, gene-weighted 2D kernel density. j , MNP-2 subset frequencies within the total T cells across all age groups including an external cord blood ( n = 3) dataset.

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: a , Overview of a fixed CITE-seq experiment ( n = 4 pediatric donors) for TCR stimulation. b , RNA-based UMAP plot of unstimulated, anti-CD3/anti-CD28 (TCR; 0.5:1 beads per cell)-stimulated and PMA/iono (PMA 50 ng ml −1 , iono 1 μg ml −1 )-stimulated pediatric CD8 + T cells. Stimulated subsets are indicated with the stimulation condition. c , Comparison of DEGs across each subset of unstimulated, TCR-stimulated and PMA/iono-stimulated CD8 + T cells. d , Violin plots of the single-cell expression of select effector genes for naive, MNP-2 and memory CD8 + T cells before and after stimulation with TCR and PMA/iono. e , Expression density of select RNA and ADT cell type markers, shown in UMAP plots of PMA/iono-stimulated and unstimulated cells. The modality of detection is indicated in square brackets. Density, gene-weighted 2D kernel density; exp, average expression level. f , Overview of an external pediatric MIS-C scRNA-seq dataset used for MNP-2 cell identification and frequency comparison. g , Frequency of MNP-2 cells in the total peripheral T cells of healthy children ( n = 6), children with active MIS-C ( n = 7) and children who had recovered from MIS-C ( n = 2).

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Comparison, Expressing

Journal: Frontiers in Neuroinformatics

Article Title: PLDAPS: A Hardware Architecture and Software Toolbox for Neurophysiology Requiring Complex Visual Stimuli and Online Behavioral Control

doi: 10.3389/fninf.2012.00001

Figure Lengend Snippet: Roadmap: an overview of the information flow within PLDAPS . In addition to the subject (which presumably has a brain), there are five components: a Plexon MAP device, the Datapixx USB peripheral, a Mac computer running Psychtoolbox, a reward dispensing system, and an input device (such as a keyboard, joystick, or eye tracker). (1) The Mac, running an experimental program in Psychtoolbox, sends digital video information to the video buffer on the Datapixx peripheral. (2) The Datapixx box splits the video information to two displays, made slightly different by two different color look-up tables (CLUT). One screen is for the subject, while the other has additional window and input markings (such as eye position) for the experimenter to monitor the subject’s activity in real-time. (3) The subject, responding to the screen, generates neural signals that are recorded by the Plexon MAP. (4) The subject also manipulates the input device. (5) Redundant analog signals are sent both to the Plexon device and the Datapixx box. (6) The Datapixx box sends the subject’s input back to the Mac, where the experimental program updates events in the trial (such as fixating on a target). (7) Timestamp markers of events in the trial are sent via TTL pulses to the Plexon MAP via an 8-bit digital cable from the Datapixx. At the end of the trial, more complex trial information is transmitted via 8-bit strobed words. (8) An analog or digital pulse is sent to the system that dispenses a reward. (9) An option is to send TTL pulses of neural spikes to the Datapixx box. This is used to closed-loop experiments where experimental parameters for the next trial are determined from neural activity in the previous trials.

Article Snippet: A central component of our system was the Datapixx multi-function data and video processing USB peripheral (VPixx Technologies).

Techniques: Activity Assay

Journal: Frontiers in Neuroinformatics

Article Title: PLDAPS: A Hardware Architecture and Software Toolbox for Neurophysiology Requiring Complex Visual Stimuli and Online Behavioral Control

doi: 10.3389/fninf.2012.00001

Figure Lengend Snippet: DIO channels for Datapixx to Plexon communication .

Article Snippet: A central component of our system was the Datapixx multi-function data and video processing USB peripheral (VPixx Technologies).

Techniques: