(A) Experimental design for the RS, RIS, CD and hTERT experiments. (B) Days in culture vs. population doublings (PDL) for WT WI-38 cells (red) and hTERT immortalized cells (green). Labeled points …
RNA-seq quality control metrics.
RNA-seq Gene expression data and differential analysis.
RNA-seq GSEA.
RNA-seq GSEA for indivdual timepoints.
Expression of canonical and novel senescence induced genes (A) RNA-seq quantification for p21, p16, NNMT, TGFB2, and GLB1 (beta-galactosidase) transcripts expressed as fold change relative to …
Senescent WI-38 cells express classic senescence markers. (A) Quantification of p16 and p14 gene products from the CDKN1A locus. Values plotted are fold change levels relative to PDL 20. TPM values …
Enrichment and induction of Epithelial to Mesenchymal Transition gene set during approach to replicative senescence. (A) GSEA was run using MSigDB Hallmark gene sets. Data used for ranked list was …
Pilot WI-38 seeding and sampling experiment. A small pilot experiment was used to determine optimal seeding and sampling cell densities in WI-38 cells at three different population doublings (PDL). …
(A) Individual UMAP projections of WT WI-38 cells; PDL colored by phases of the cell cycle (G1 = green, G2/M = orange, S-phase = purple). (B) Bar graph of cell cycle state percentages defined by …
Using a modified resuspension buffer of DMEM + FBS increases detection of senescent cells with the 10 x Genomics 3’ single cell RNA-seq protocol. (A) Barplot comparing the number of cells detected …
Replicative senescence dependent gene expression changes measured by Bulk RNA-seq and single cell RNA-seq are highly concordant.
Scatterplot comparing the log2 fold changes of PDL 50 vs. PDL 25 measured using single-cell pseudo-bulk count summations (x-axis) and actual bulk RNA-seq (y-axis). The color for each gene is the log10 normalized counts. The r for all genes is 0.77. The r for genes filtered at >25 th percentile is 0.81 (red line) and 0.45 for genes < 25 th percentile (blue line).
Single cell UMAPs of high mitotic scoring (gene set) WI-38 cells. S phase and G2M scored cells were separated and reprocessed and visualized with a UMAP projection. In mitotic cells, PDL is the …
WT and hTERT WI-38 cells scored for senescence and cell density signatures. (A) UMAP projection of WT and hTERT single-cell RNA-seq scored with a signature composed of significantly increasing genes …
Representative images of WI-38 cells at collection time points.
(A) Significant (Benjamini-Hochberg adjusted pvalue < 0.01) GSEA results for protein changes (n=3) using the MSigDB Hallmarks. Reference time point not shown. Values are -log10 p-value and are …
Proteomics quantification, differential analysis and GSEA.
Metabolomics quantification and differential analysis.
Heatmap of hierarchical clustering of 8000 protein log2 fold changes at each time point/PDL versus first (not shown) for RS WT WI-38 (left) and hTERT WI-38 cells (right) from high induction (orange) …
Scatterplot comparing the log2 fold change expression of genes (x-axis) vs. protein (y-axis) in PDL 50 cells relative to PDL 20 cells. Oxidative phosphorylation leading edge genes from GSEA plotted …
Breakdown of the hallmark oxidative phosphorylation gene set into functional subsets reveals up regulation of most mitochondrial functions during replicative senescence. Heatmaps of log2 fold change …
PDL-dependent changes in the senescent metabolome vs. hTERT cells. Heatmap of hierarchical clustering of 285 metabolite log2 fold changes at each PDL or PDL.ctrl versus first (not shown) for RS WT …
Metabolites in blue, proteins in green. Heatmaps of log2 fold changes for metabolites and proteins from A. Median hTERT corrected values for n=4 replicates are shown. Significant changes (FDR …
(A) Gene expression fold changes for the NNMT (top) in the hTERT, replicative senescence (RS), radiation induced senescence (RIS), and cell density (CD) time courses (three replicate average, each …
(A) Percent of ATAC-seq reads falling into four broad chromatin states compiled from the ENCODE IMR-90 25 chromatin state prediction for all samples in WT or hTERT WI-38 cells. Percent of all …
ENCODE IMR90 chromatin state labels.
ATAC-seq peak coordinates and metadata.
Normalized ATAC-seq coverage in peaks.
ATAC-seq fraction of reads in peaks.
IMR90 LADs from Salama 2013.
IMR90 NADs from Nemeth 2017.
ATAC-seq library fragment distribution and size selection.
ATAC-seq library fragment distribution after size selection and sequencing.
ATAC-seq mitochondrial read percentages and ATAC-seq transcriptional start site enrichment. (A) Mitochondrial read percentages in sequenced and aligned ATAC-seq library for WT PDLs (red) hTERT time …
ATAC-seq QC metrics, controls, and NADs/LADs browser shot. (A) Fraction of reads in peak by PDL (WT) and PDL.ctrl (hTERT).p values for linear fit over time are shown. WT cells exhibit a slight ~5% …
Chromatin state profiles of ATAC-seq peaks in NADs vs. rest of the genome. Heatmap of all significant ATAC-seq peaks categorized by 25 chromatin states and then divided into peaks overlapping with …
(A) Scatter plot of transcription factor motif enrichment in ATAC-seq peaks surrounding significantly induced genes (FDR adjusted p-value < 0.01, log2 FC >0.5) during replicative senescence. The …
Gene expression fold changes for the transcription factor FOXE1 across conditions. Gene expression fold changes (three replicate average, each point a replicate) for the transcription factor FOXE1 …
(A) UMAP projection of single WI-38 cells collected at increasing PDLs (PDL25-red) to (PDL50-blue) and colored by pseudotime (top). (B) Scatterplot of single-cell gene expression across pseudotime …
Gene expression changes with pseudotime.
GO term enrichment in pseudotime clusters.
Heatmap of gene expression changes for the top 5000 significantly changing genes during replicative senescence pseudotime. Genes were hierarchically clustered, gene normalized and scaled (0–1) …
Heatmap of enriched gene sets for replicative senescence pseudotime clusters (k-25). Heatmap of adjusted p-values for GO-term enrichment analysis on individual clusters from 6. Values plotted are …
(A) RNA-seq and proteomics heatmaps of selected genes based on myofibroblast markers and biology. Values plotted are log2 fold change of each PDL vs. PDL 20 for replicative senescence timecourse and …
RNA-seq data for verteporfin treatment.
Effect of inhibiting the YAP1/TEAD1 interaction with verteporfin treatment on WI-38 cells. (A) Barplots of the gene expression of 3 known YAP1 gene targets in verteporfin-treated cells. Values …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell Line (H. sapiens) | WI-38 fibroblasts | Coriell | AG06814-N | |
Cell Line (H. sapiens) | WI-38 hTERT | This paper | immortalized WI-38 human fibroblasts, methods | |
Transfected construct (H. sapiens) | pCDH-CMV-hTERT-EF1a-puro | This paper | Lentiviral plasmid, methods | |
Commercial assay, kit | Senescence-β-Galactosidase | Cell Signaling Tech. | 9,860 | |
Commercial assay, kit | MycoAlert Mycoplasma Detection | Lonza | LT07-218 | |
Commercial assay, kit | Direct-zol RNA Miniprep Plus | Zymo Research | R2072 | |
Commercial assay, kit | Chromium Single Cell 3’ v2 | 10 x Genomics | 120,237 | |
Commercial assay, kit | Chromium Single Cell A Chip | 10 x Genomics | 1000009 | |
Commercial assay, kit | Tagment DNA Enzyme and Buffer | illumina | 20034197 | |
Commercial assay, kit | Clean-and-Concentrator-5 | Zymo Research | D4014 | |
Commercial assay, kit | NEBNext High-Fidelity 2 X PCR | NEB | M0541L | |
Commercial assay, kit | TruSeq Stranded mRNA Library | illumina | 20020595 | |
Commercial assay | Bioanalyzer High Sensitivity DNA | Agilent | 5067–4626 | |
Commercial assay, kit | Pierce BCA Protein Assay | Thermo Fisher | 23,227 | |
antibody | anti-human p16 antibody (Mouse monoclonal) | BD Biosciences | RRID:AB_395229 | (1:250) |
antibody | anti-human p21 antibody (Mouse monoclonal) | BD Biosciences | RRID:AB_396414 | (1:250) |
Commercial assay, kit | Pippin Prep 2% 100–600 bp | Sage Science | CDF2010 | |
Chemical compound, drug | Verteporfin | RD Systems | 1243926 | |
Other | Zorbax Extend C18 column | Aglient | 759700–902 | |
Other | SeQuant ZIC-pHILIC column | EMD Millipore | 150,460 | |
Software, algorithm | R (v4.0.3 and 3.6.2) | r-project.org/ | RRID:SCR_001905 | |
Software, algorithm | Salmon (v 0.8.2) | combine-lab.github.io/salmon/ | RRID:SCR_017036 | |
Software, algorithm | DESeq2 (v1.30.1) | bioconductor | RRID:SCR_015687 | |
Software, algorithm | sva package(v3.38.0) | bioconductor | RRID:SCR_012836 | |
Software, algorithm | fgsea 1.16.0 | bioconductor | RRID:SCR_020938 | |
Software, algorithm | CellRanger 3.0 | 10 x Genomics | RRID:SCR_017344 | |
Software, algorithm | SCTransform (v 0.3.2) | Satijalab.org/seurat | ||
Software, algorithm | Seurat (v4.0.1.9005) | satijalab.org/seurat | RRID:SCR_007322 | |
Software, algorithm | monocle3 (v1.0.0) | cole-trapnell-lab.github.io/monocle3 | RRID:SCR_018685 | |
Software, algorithm | bowtie2 (v2.3.4.1) | bowtie-bio.sourceforge.netbowtie-bio.sourceforge.net | RRID:SCR_016368 | |
Software, algorithm | samtools (v1.2) | http://ww.htslib.org/ | RRID:SCR_002105 | |
Software, algorithm | Picard (v2.6.4) | broadinstitute.github.io/picard | RRID:SCR_006525 | |
Software, algorithm | macs2 (v2 2.1.2) | hbctraining.github.io/main/ | RRID:SCR_013291 | |
Software, algorithm | GenomicRanges (v1.42.0) | bioconductor | RRID:SCR_000025 | |
Software, algorithm | cutadapt (v2.4) | https://github.com/marcelm/cutadapt | RRID:SCR_011841 | |
Software, algorithm | bcl2fastq (v2.20) | Illumina | RRID:SCR_015058 | |
Software, algorithm | regioneR v1.22.0 | bioconductor | ||
Software, algorithm | ATACseqQC v1.14.4 | bioconductor | ||
Software, algorithm | LIMMA v3.46.0 | bioconductor | RRID:SCR_010943 | |
Software, algorithm | Qvalue v2.26.0 | combine-lab.github.io/salmonQvalue | RRID:SCR_001073 | |
Software, algorithm | LISA v1 | lisa.cistrome.org |