Single-cell transcriptomics of X-ray irradiated Drosophila wing discs reveals heterogeneity related to cell-cycle status and cell location
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
Figures
Figure 1
Effects of X-ray irradiation on DNA damage, apoptosis, and cell cycle progression.
(A) Cartoon overlay of wing disc showing proximodistal (PD) regions. (B, C) Immunofluorescence (IF) of p-H2Av at 0 rad (B) and 4000 rad of irradiation, 30 min after exposure (C). (D, E) IF of cleaved Dcp-1 at 0 rad (D) and 4000 rad 4 hr after exposure (E). Magenta arrowhead points to the DV boundary and white arrowheads point to regions of the hinge with less Dcp-1 signal. (F–I) IF of PHH3 at 0 rad (F) and 4000 rad 15 min (G), 2 h (H), and 4 hr (I) after exposure. (J–M) EdU incorporation at 0 rad (J) and 4000 rad 15 min (K), 2 h (L), and (M) 4 hr after exposure. Magenta arrowheads point to regions with high EdU signal at 4 hr after irradiation. All scale bars are 100 μm. For B–M, DAPI is in blue.
Figure 2 with 1 supplement
Territories of the wing disc shown by immunofluorescence and UMAP plots.
Immunofluorescence (IF) of Zfh2 (A, B), GFP expression in w-, nub-gal4, uas-GFP; fly wing discs (A’, B’), and merged images of both (A’’, B’’) at 0 rad (A–A’’) or 4 hr after exposure to 4000 rad (B–B’’). For A–B’, DAPI is in blue. All scale bars are 100 μm. (C) UMAP showing 35 cluster annotations with cluster annotations based on proximodistal (PD) region. (D) UMAP showing broad epithelial region annotations. (E–H) UMAP plots showing the expression of markers used to annotate specific epithelial regions at 0 rad (top row) and 4000 rad (bottom row); Ubx for PE (E), tup for notum (F), zfh2 for hinge (G), nub for pouch (B). Combinations were used to determine hinge-notum and hinge-pouch regions. Plots for eyg (also used for the notum) and twi (used for the myoblasts) are shown in Figure 2—figure supplement 1.
Figure 2—figure supplement 1
UMAP plots of top cluster markers by region.
(A) Expression UMAPs of eyg at 0 rad (left) and 4000 rad (right). (B) Expression UMAPs of twi at 0 rad (left) and 4000 rad (right). (C) Cropped and zoomed-in UMAP plots of clusters belonging to each of the major proximodistal (PD) regions. Each cluster is labeled by its cluster number and the genes that are most highly expressed in it when compared to all clusters (left of the slash) and when compared to other clusters within the same PD region (right of the slash).
Figure 3 with 6 supplements
Heterogeneity of gene expression across the wing disc following radiation exposure.
(A) 521 genes across nine functional categories plotted as points by their HHI score at 4000 rad. Point color represents the log2FC between conditions in the cluster that has the highest log2FC. The horizontal red bar represents the mean HHI score for the genes of that category. Genes with max log2FC >2 or HHI >0.075 were labeled where space permitted. The equation used to calculate the Herfindahl-Hirschman Index (HHI) is shown above the panel. (B) Heat map of four highest HHI scoring genes in apoptosis, DNA damage response (DDR), reactive oxygen species (ROS), cell cycle categories (low HHI scoring categories), and transcription factor (TF) and ligand categories (categories with high HHI scoring genes). Box colors represent the proportion of mRNA found in each subregion relative to the sum of all mRNA found in all clusters (the values used to calculate HHI). (C) X-ray induced genes plotted by their HHI score (calculated on seven broad regions, not the 35 clusters) and the Euclidean distance in gene expression in seven-dimensional space at 0 rad vs 4000 rad (calculated using the seven broad regions as described in the text). Point color represents the log2FC of each gene when comparing all cells from the 4000 rad condition to all cells of the 0 rad condition. The dotted lines are drawn at the 95th percentile for each of the two parameters. (D–G) Gene expression UMAPs of example genes with <95 th percentile Euclidean distance score belonging to the DDR (D), apoptosis (E), ROS (F), and ligand (G) groups. (H–I) Gene expression UMAPs of two genes with large differences in expression pattern between 0 rad and 4000 rad identified by a top 5% Euclidean distance. Genes in (D–I) are arranged from high (top) to low (bottom) HHI score.
Figure 3—figure supplement 1
Histogram of cluster Herfindahl-Hirschman Index (HHI) scores, broad region HHI scores, and Euclidean expression distance scores.
(A) Histogram of cluster HHI scores for 3767 X-ray induced genes that meet the following criteria: Adjusted p-value <0.05, average log2FC >0.1, percent of cells expressing the gene in either condition ≥0.01. The inset is the same histogram cropped on the y-axis to better view the distribution. (B) Histogram of broad region HHI scores of 3655 X-ray induced genes that meet the following criteria: Adjusted p-value <0.05, Average log2FC >0.1, Percent of cells expressing the gene in both conditions ≥1%. The inset is the same histogram cropped on the y-axis to better view the distribution. (C) Histogram of Euclidean expression distances for the same 3655 genes as in (B). The inset is the same histogram cropped on the y-axis to better view the distribution.
Figure 3—figure supplement 2
Proportion mRNA heatmaps of ligands and transcription factors.
Heatmaps showing the proportion of mRNA found in each of the 35 clusters in the 4000 rad condition for all genes in the ligand (A) and transcription factor (TF) (B–B’) gene categories.
Figure 3—figure supplement 3
Proportion mRNA heatmaps transcription factors continued.
Heatmaps showing the proportion of mRNA found in each of the 35 clusters in the 4000 rad condition for transcription factors (TFs) continued (A–A’).
Figure 3—figure supplement 4
Proportion mRNA heatmaps of apoptosis, reactive oxygen species (ROS), cell cycle, and DNA damage response (DDR) genes.
Heatmaps showing the proportion of mRNA found in each cluster in the 4000 rad condition for all genes in the Apoptosis (A) ROS (B), cell cycle (C), and DDR (D) gene categories.
Figure 3—figure supplement 5
Proportion mRNA heatmaps of kinase, phosphatase, and receptor genes.
Heatmaps showing the proportion of mRNA found in each cluster in the 4000 rad condition for all genes in the kinase (A–A’), phosphatase (B), and receptor (C) gene categories.
Figure 3—figure supplement 6
Examples of genes spanning a range of Euclidean expression distance scores.
(A) Heat map showing the difference in the proportion of gene expression in each proximodistal (PD) region between 4000 and 0 rad. Numbers in the tiles are the values of this difference with positive values indicating a higher proportion at 4000 rad for a given region, and negative values indicating a lower proportion at 4000 rad. For example, a value of 0.4 means that the proportion of expression in a given region at 4000 rads was 0.4 greater than at 0 rads, and a value of –0.4 means that it is 0.4 less. Euclidean distance was calculated on the absolute values of these differences. Genes were selected to span the range of Expression Distances. (B) Plot showing the distribution of Euclidean expression distances for the 3655 genes plotted in Figure 3C. The dotted line is drawn at the 95th percentile of Euclidean expression distance scores. Genes from panel A are highlighted. (C) Expression UMAPs of genes spanning the range of Euclidean expression distance scores, ordered in ascending order of Euclidean expression distance (number above UMAPs). The dotted line separates genes below (left of the line) and above (right of the line) the 95th percentile.
Figure 4 with 2 supplements
X-ray-induced expression of genes encoding ligands.
(A) Heatmaps showing log2FC between conditions for each cluster (left) and proportion of total mRNA in each cluster at 4000 rad (right) of the top 14 ligands ranked by max log2FC in any cluster. A Left: Boxes are colored based on the average log2FC from 0 rad to 4000 rad for each cluster. The left number in each box is the percentage of cells in the cluster expressing at least one transcript at 0 rad; the right number is the same at 4000 rad. Dark black numbers indicate that the change is statistically significant in that cluster (adjusted p-value <0.05, Wilcoxon Rank Sum Test, Bonferroni correction), while light gray numbers indicate that it is not (adjusted p-value ≥0.05). Genes are sorted from left to right in descending order of the mean log2FC when comparing all cells of 0 rad to all cells of 4000 rad (‘overall mean log2FC’); the overall mean log2FC value is in parentheses next to gene names (all significant). A Right: Boxes are colored based on the proportion of mRNA expressed in that cluster vs the total amount of mRNA expressed in all clusters. Genes are sorted from left to right in ascending order of Herfindahl-Hirschman Index (HHI) score. HHI score is noted in parentheses next to gene name. Genes shown in panels (B–G) are underlined with gray bars in (A). (B–M) In each panel: hybridization chain reaction (HCR) signal of upd1, upd2, and upd3 are in yellow and DAPI in blue at 0 rad (B, D, F, H, J, L) or 4000 rad (C, E, G, I, K, M). Wild-type discs (B, C, F, G, J, K) are compared to p53 mutant discs (D, E, H, I, L, M). To the right of each wild-type HCR image is an expression UMAP of each gene in each condition. The myoblast cluster was cropped into UMAP images for space, indicated by the dotted box around them.
Figure 4—figure supplement 1
Toll and PDGF/VEGF ligands and receptors.
Expression UMAPs of Toll receptors (A), Toll ligands (B), PDGF/VEGF receptors (C), and PDGF/VEGF ligands (D).
Figure 4—figure supplement 2
JAK/STAT receptors and tumor necrosis factor (TNF) ligands and receptors.
Expression UMAPs of JAK/STAT receptors (A), TNF receptors (B), and TNF ligands (C).
Figure 5 with 1 supplement
X-ray induced expression of transcription factors.
(A) Heatmaps showing log2FC between conditions for each cluster (left) and proportion of total mRNA in each cluster at 4000 rad (right) of the top 14 transcription factors (TFs) ranked by max log2FC in any cluster. Generated in the same way as Figure 4A. Genes shown in panels (B–T) are underlined with gray bars in (A). (B, G, L, Q, S) hybridization chain reaction (HCR) of p53, Ets21C, dysf, Xrp1, and Dif at 0 rad in Oregon R wing discs. (C, H, M, R, T) HCR of the same genes at 4000 rad in Oregon R wing discs. For B–T, HCR signal is in yellow and DAPI in blue. To the right of 0 rad and left of 4000 rad HCR image is an expression UMAP of each gene in each condition. (D, I, N) Alternative irradiated wing disc showing non-induction of dysf after X-ray exposure. The myoblasts were cropped into the UMAP images for space, indicated by the dotted box around them. (E, J, O) HCR of p53, Ets21C, and dysf in p53[5 A-1-4] mutant wing discs at 0 rad. (F, K, P) HCR signal of the same genes at 4000 rad in p53[5 A-1-4] mutant wing discs.
Figure 5—figure supplement 1
Expression UMAP of Toll transcription factor (TF), high magnification images of dysf hybridization chain reaction (HCR), and increased intensity images of Xrp1 and p53.
(A) Heatmap of the Toll TF Dl. (B) 63 X magnification images of dysf HCR. White arrowheads point to the location of normal developmental expression of dysf in the posterior hinge (pouch is below this location). Magenta arrowheads point to X-ray induced dysf mRNA which is localized to the nucleus. Scale bars are 50 μm. (C) HCR of p53 and Xrp1 with increased gain. For B and C, HCR signal is in yellow and DAPI is in blue. Images in each row have the same min/max display. Scale bars are 100 μm.
Figure 6 with 3 supplements
Clustering of cells on 175 cell cycle genes.
(A–A’) Cluster UMAPs of data processed and clustered on cell cycle genes at 0 rad (A) and 4000 rad (A’). (B–C’) Expression of trbl and PCNA in this UMAP object at 0 rad (B, C) and 4000 rad (B’, C’). (D) Heatmap showing average scaled expression of cell cycle marker genes in each cell cycle cluster considering both integrated conditions. Numbers are the percent of cells expressing each gene in each cluster. (E) Bar plot showing the proportion of cells each cluster contributes to total cells at 0 rad (cream) and total cells at 4000 rad (teal) conditions. (F–H’) High-trbl cluster (in orange) and both high-PCNA clusters (in purple) from panels A-A’ mapped onto the standard UMAP at 0 rad (F) and 4000 rad (F’). (G–H’) Standard UMAPs showing expression of PCNA and trbl at 0 rad (G, H) and 4000 rad (G’, H’).
Figure 6—figure supplement 1
Cluster tree showing cluster stability at different clustering resolutions.
Tree showing clusters generated using different Louvain clustering resolution parameters on the integrated principal component dimensions derived from the 175 cell cycle genes. Each node is a cluster (clusters are numbered in descending order of cluster size but the numbering is otherwise arbitrary). Each row of clusters from top to bottom are derived from increasing resolutions. Arrows represent cells moving from one cluster identity at a lower resolution to a different cluster identity at a higher resolution. Arrow color represents the number of cells making the transition, and arrow transparency represents the proportion of contribution of those cells to the new cluster.
Figure 6—figure supplement 2
Dendrogram of cell-cycle-based cluster relationships.
Dendrogram showing the relationship of cell-cycle-based clusters to one another in integrated PC space generated from 175 cell-cycle genes. Shorter lines represent a closer relationship; longer lines represent a more distant relationship.
Figure 6—figure supplement 3
Expression of cell-cycle-based cluster markers.
(A–B) Anti-GFP staining in unirradiated (A) and irradiated trbl-GFP wing discs (B). DAPI is blue and anti-GFP is yellow. (C–H) Hybridization chain reaction (HCR) of trbl, PCNA, and bora in unirradiated (C–E) and irradiated (F–H) Oregon R wing discs. HCR signal is yellow and DAPI is blue. (I) Quantification of trbl, PCNA, and bora HCR signal in unirradiated and irradiated discs. Intensity measurements were the mean intensity of each gene in the entire disc. For each gene, values were normalized to the mean intensity of the 0 rad condition. Two-tailed Welch’s t-tests were used to compare the values of each gene in irradiated to unirradiated conditions. All quantification was performed on max projections. P-values are above each comparison.
Figure 7 with 2 supplements
Differences in X-ray induced gene expression between cell-cycle-based clusters.
(A) Mean scaled expression of X-ray induced genes with avg log2FC ≥1 between conditions. Mean-scaled expression values were calculated from cell-cycle-based clusters using cells from the 4000 rad condition only. Each row contains the same genes. (B) Heatmap showing the average scaled expression of the top 24 X-ray-induced genes with highest expression in the High-trbl Cluster. (C) 24 of the genes with maximum expression in a cell-cycle-based cluster other than the High-trbl Cluster. Numbers are the percentage of cells expressing the gene in each cluster. Only genes with ≥5% expression in any cluster at 4000 rad were selected from the initial 359.
Figure 7—figure supplement 1
Effects of trbl overexpression on cell death.
(A) Schematic of timed overexpression in wing discs. GFP or trbl were driven for 24 hr prior to irradiation and during recovery in the pouch using rn-gal4 and gal80ts. (B–E) Dcp-1 antibody staining of unirradiated discs overexpressing GFP (B) or trbl (C) and irradiated discs expressing GFP (D) or trbl (E). Dcp-1 is yellow and DAPI is blue. (F) Quantification of the mean Dcp-1 signal in the inner-pouch of discs (the region within the fold nearest to the center of the pouch) from each condition in B–E. Values were normalized to the mean Dcp-1 signal in the rnts >GFP, 0 rad condition. Two-tailed Welch’s t-tests were used to compare Dcp-1 signal in rnts >trbl (n=2) to rnts >GFP (n=6) samples in the 0 rad condition and to compare rnts >trb (n=9) to rnts >GFP (n=8) samples in the 4000 rad condition. All quantification was performed on max projections. P-values are above each comparison.
Figure 7—figure supplement 2
Effects of trbl overexpression on HIX genes Swim and CG15784.
(A–D) Hybridization chain reaction (HCR) of trbl (magenta) in unirradiated rnts >GFP (A) or rnts >trbl wing discs (B) and irradiated rnts >GFP (C) or rnts >trbl discs (D). (E–H) HCR of Swim (yellow) in the same unirradiated rnts >GFP (E) or rnts >trbl discs (F) and the same irradiated rnts >GFP (G) or rnts >trbl discs (H). (I) Quantification of Swim in the pouch of discs from each condition of (A–D). Values were normalized to the mean Swim signal in the rnts >GFP, 0 rad condition. Two-tailed Welch’s t-tests were used to compare the Swim signal between irradiated and unirradiated rnts >GFP discs and to compare the Swim signal between irradiated and unirradiated rnts >trbl discs. All quantification was performed on max projections. P-values are above each comparison. (J–M) HCR of trbl (magenta) in unirradiated rnts >GFP (J) or rnts >trbl wing discs (K) and irradiated rnts >GFP (L) or rnts >trbl discs (M). * Next to K: in K, trbl signal was adjusted for brightness independently of J, L, and M, as this condition had a relatively weak trbl signal. (N–R) HCR of CG15784 (yellow) in the same unirradiated rnts >GFP (N) or rnts >trbl discs (O) and the same irradiated rnts >GFP (P) or rnts >trbl discs (Q). For A–Q, DAPI is in blue. (R) Quantification of CG15784 signal in the pouch of discs from each condition of (A–D). Values were normalized to the mean CG15784 signal in the rnts >GFP, 0 rad condition. Two-tailed Welch’s t-tests were used to compare the CG15784 signal between irradiated and unirradiated rnts >GFP discs and to compare the CG15784 signal between irradiated and unirradiated rnts >trbl discs. All quantification was performed on max projections. P-values are above each comparison.
Figure 8
Herfindahl-Hirschman Index (HH)I scoring of categorical genes in standard clusters vs cell-cycle-based clusters.
(A–I) Transformed HHI scores calculated using the 35 standard clusters (y-axis) and the six cell-cycle-based clusters (x-axis). Genes used were the 521 categorical genes shown previously in Figure 3A. Scores were calculated using cells from the 4000 rad condition only. Genes displayed encode for proteins involved in apoptosis (A), DNA damage response (DDR) (B), reactive oxygen species (ROS) (C), cell cycle (D), transcription factors (TFs) (E), phosphatases (F), kinases (G), ligands (H), and receptors (I). Genes are labeled where space permits but were otherwise chosen for labeling arbitrarily.
Figure 9
Two types of heterogeneity following X-ray irradiation.
(A) Inter-regional heterogeneity. Many genes are expressed uniformly while some are expressed in specific territories following irradiation. (B) Intra-regional heterogeneity. In the example shown, a gene is expressed at high levels in some cells, intermediate levels in some, and no expression in others. Cells with different levels of expression are interspersed.
Author response image 1
RNA in situ hybridizations using the hybridization chain reaction performed using probes to trbl.
In A-F, the RNAi is expressed using nubbin-Gal4. In G-I the RNAi is expressed using rn-Gal4, tub-Gal80ts. white-RNAi was used as a control (A, B, G, H). Three different RNAi lines directed against trbl were tested: Vienna lines VDRC 106774 (C, D) and VDRC 22113 (E, F), and Bloomington line BL42523. In no case was a reduction in trbl RNA upregulation in the wing pouch following 4000 rad observed, except for one disc (n = 6) of VDRC 106774 crossed to nubbin-gal4.
Author response image 2
Images for discs shown in Manuscript Figure 1panels B, C.
Author response image 3
Images for discs shown in Manuscript Figure 1panels D, E.
Author response image 4
Images used in Manuscript Figure 1, F, G.
Author response image 5
Images used in Manuscript Figure 1H, I.
Author response image 6
Images used in Manuscript Figure 1J, K.
Author response image 7
Images used in Manuscript Figure 1L, M.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | Oregon R | Bloomington Drosophila Stock Center | Stock #25211; RRID:BDSC_25211 | |
| Strain, strain background (Drosophila melanogaster) | p53[5 A-1-4] | Bloomington Drosophila Stock Center | Stock #6815; RRID:BDSC_6815 | |
| Strain, strain background (Drosophila melanogaster) | UAS-trbl | Bloomington Drosophila Stock Center | Stock #58493; RRID:BDSC_58493 | |
| Strain, strain background (Drosophila melanogaster) | UAS-GFP | Bloomington Drosophila Stock Center | Stock #4776; RRID:BDSC_4776 | |
| Strain, strain background (Drosophila melanogaster) | trbl-GFP | Bloomington Drosophila Stock Center | Stock #61654; RRID:BDSC_61654 | |
| Antibody | Rabbit anti-H2AvD-pS13 (polyclonal) | Rockland | Cat #600-401-914; RRID:AB_828383 | (Dilution used = 1:250) |
| Antibody | Rabbit anti-Dcp-1 (polyclonal) | Cell Signaling | Cat #9578; RRID:AB_2721060 | (Dilution used = 1:250) |
| Antibody | Rabbit anti-PHH3 (polyclonal) | Millipore | Cat #06–570; RRID:AB_310177 | (Dilution used = 1:500) |
| Antibody | Rat anti-Zfh2 (polyclonal) | Chris Doe; Tran et al., 2010 | https://doi.org/10.1242/dev.048678 | (Dilution used = 1:200) |
| Antibody | Rabbit anti-GFP (polyclonal) | Torrey Pines Biolabs, Inc | Cat #TP401 | (Dilution used = 1:500) |
| Antibody | Goat anti-rat Alexa Fluor 555 (polyclonal) | Thermo Fisher Scientific | Cat #A-21434; RRID:AB_2535855 | (Dilution used = 1:500) |
| Antibody | Goat anti-rabbit Alexa Fluor 555 (polyclonal) | Thermo Fisher Scientific | Cat #A-21428; RRID:AB_2535849 | (Dilution used = 1:500) |
| Commercial assay, kit | HCR RNA-Fish (v3) (buffers, hairpins, probes) | Molecular Instruments | ||
| Commercial assay, kit | Click-iT EdU Cell Proliferation Kit, Alexa Fluor 647 | Thermo Fisher Scientific | Cat #C10340 | |
| Software, algorithm | Image J / Fiji | https://fiji.sc/ | ||
| Software, algorithm | Kallisto-Bustools (v0.28.2) | Pachter Lab | https://www.kallistobus.tools/ | |
| Software, algorithm | R | R Project for Statistical Computing | http://www.r-project.org/ | |
| Software, algorithm | org.Dm.eg.db (v3.18.0) | Bioconductor | https://www.bioconductor.org/packages/release/data/annotation/html/org.Dm.eg.db.html | |
| Software, algorithm | Seurat (v5) | Comprehensive R Archive Network | https://cran.r-project.org/web/packages/Seurat/index.html | |
| Software, algorithm | clustree | Comprehensive R Archive Network | https://cran.r-project.org/web/packages/clustree/index.html |
Additional files
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Supplementary file 1
Top markers for each cluster.
Contains the following columns (Column; description): Subregion; cluster name Marker_Integrated_Global; Top marker gene when comparing cluster to all other cells in the integrated data. Pct_Diff_Integrated_Global; Percent of cells expressing top marker in cluster of interest minus percent of cells expressing in all other cells in the integrated data. Marker_R4K_Global; Top marker gene when comparing cluster to all other clusters in the 4000 rad condition. Pct_Diff_R4K_Global; Percent of cells expressing top marker in cluster of interest minus percent of cells expressing in all other cells in the 4000 rad condition. Marker_R0K_Global; Top marker gene when comparing cluster to all other clusters in the 0 rad condition. Pct_Diff_R0K_Global; Percent of cells expressing top marker in cluster of interest minus percent of cells expressing in all other cells in the 0 rad condition. Marker_Integrated_Region; Top marker gene when comparing cluster to all other clusters within its broad PD region in the integrated data. Pct_Diff_Integrated_Region; Percent of cells expressing top marker in cluster of interest minus percent of cells expressing in all other cells within its broad PD region in the integrated condition. Marker_R4K_Region; Top marker gene when comparing cluster to all other clusters within its broad PD region in the 4000 rad condition. Pct_Diff_R4K_Region; Percent of cells expressing top marker in cluster of interest minus percent of cells expressing in all other cells within its broad PD region in the 4000 rad condition. Marker_R0K_Region; Top marker gene when comparing cluster to all other clusters within its broad PD region in the 0 rad condition. Pct_Diff_R0K_Region; Percent of cells expressing top marker in cluster of interest minus percent of cells expressing in all other cells within its broad PD region in the 0 rad condition. Markers were only considered if (1) they were expressed in at least 10% of cells in the cluster being considered or its comparison group, (2) there was a minimum difference of 10% between the cluster being considered or its comparison group, and (3) there was an enrichment of at least log2FC = 1 in the cluster being considered. Ties were broken by taking the marker with the higher log2FC.
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp1-v1.csv
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Supplementary file 2
DEG 4000 rad vs 0 rad.
Contains the following columns (Column; description): gene_name; Gene name. p_val; unadjusted p-value of Wilcoxon rank-sum test. avg_log2FC; average log2FC of all cells in 4000 rad condition versus all cells in 0 rad condition. pct.1; Percent of cells expressing given gene in 4000 rad condition. pct.2; Percent of cells expressing given gene in 0 rad condition. p_val_adj; Bonferroni corrected p-value from p_val column (corrected on total genes captured = 13,384). Genes were only included in this table if they were present in at least 1% of cells in either condition, had an adjusted P-value <0.05, and had an average log2FC of ≥0.1. This table was produced using the FindMarkers() function in Seurat v5.
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp2-v1.csv
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Supplementary file 3
Apoptosis, DNA damage response (DDR), response to reactive oxygen species (ROS), cell cycle regulation, transcription factors (TFs), phosphatases, kinases, ligands, and receptors genes considered for HHI comparison (pre-filter).
Contains the following columns (Column; description): genes_name; All genes captured in this data belonging to the considered categories, 1732 total. Category; Category each gene is classified into.
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp3-v1.csv
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Supplementary file 4
Genes from Supplementary file 3 with more than one category and their placements.
Contains 81 genes that were found in more than one of the nine categories in Supplementary file 3. Contains the following columns (Column; description): genes_name; Genes from Supplementary file 3 that were initially found in more than one category. Categories; Categories gene was found in. Category; Category each gene was chosen to be classified into (these are the classifications used in Supplementary file 3).
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp4-v1.csv
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Supplementary file 5
Apoptosis, DNA damage response (DDR), response to reactive oxygen species (ROS), cell cycle regulation, transcription factors (TFs), phosphatases, kinases, ligands, and receptors genes considered for HHI comparison (post-filter, n=521).
Contains the following columns (Column; description): gene_name; Gene name. Includes all 521 genes used in Figures 3A and 8. HHI_R0K; HHI score in the 0 rad condition. HHI_R4K; HHI score in the 4000 rad condition. p_val; p-value of Wilcoxon rank sum test for cluster of maximum FC. avg_log2FC; Log2FC between conditions of the cluster with the highest log2FC. pct.1; Percent of cells cluster of highest log2FC in the 4000 rad condition. pct.2; Percent of cells cluster of highest log2FC in the 0 rad condition. p_val_adj; Bonferroni adjusted p-value from p_val column (note only corrected for genes, not number of cluster FC tests ran). Cluster_max_FC; Cluster with the highest FC. Group; Gene category.
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp5-v1.csv
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Supplementary file 6
Cell cycle gene markers for cell-cycle-based clusters.
Contains the following columns (Column; description): gene; Gene name. avg_scaled_…; Average scaled expression in each cell cycle cluster (integrated). 6 columns. Percent_Expressed_…; Percentage of cells expressing given gene in each cell cycle cluster (integrated). 6 columns. This table includes all 175 genes used in cell-cycle-based clustering.
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp6-v1.csv
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Supplementary file 7
Highly induced gene markers for cell-cycle-based clusters.
This table includes the 359 genes that are highly induced after irradiation. These genes are expressed in at least 1% of cells in either condition, have an average log2FC ≥1, and an adjusted P-value <0.05. Contains the following columns (Column; description): gene; Gene name. avg_scaled_…; Average scaled expression in each cell cycle cluster in the 4000 rad condition. 6 columns. Percent_Expressed_…; Percentage of cells expressing given gene in each cell cycle cluster in the 4000 rad condition. 6 columns.
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp7-v1.csv
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Supplementary file 8
Table containing FlyBase gene IDs, FlyBase gene CGs, and current gene symbols used in org.Dm.eg.db v3.18.0 R package to translate FlyBase IDs to Symbols.
This table contains a list of 25,107 genes identified by their Flybase IDs, CGs, and current symbols at the time of analysis used to convert FlyBase IDs to Symbols. Note, not all genes were captured in this dataset. Contains the following columns (Column; description): FLYBASE; FlyBase ID. FLYBASECG; FlyBase CG. SYMBOL; Symbol.
- https://cdn.elifesciences.org/articles/106410/elife-106410-supp8-v1.csv
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MDAR checklist
- https://cdn.elifesciences.org/articles/106410/elife-106410-mdarchecklist1-v1.docx
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Single-cell transcriptomics of X-ray irradiated Drosophila wing discs reveals heterogeneity related to cell-cycle status and cell location
eLife 14:RP106410.
https://doi.org/10.7554/eLife.106410.3
