EMBO/SourceData huggingface dataset:
Figure 2 B. Immunoblot analysis (IB) of GST-pulldown samples with deletion mutants of FUS‑NTD‑GST as baits with/without cell extract (C.E). ARID1A and BRG1 were used as tracers of SWI/SNF binding. Coomassie staining of recombinant proteins are shown in the lower panel. Amino acid numbers are indicated  C. Immunoblot analysis (IB) of FUS-NTD‑GST pulldown samples of cell extracts (C.E.) after treatment with RNase or DNase, using antibodies against ARID1A and BRG1  D. Immunoblot analysis (IB) of SWI/SNF components (ARID1A, BRG1, BAF170, BAF155, BAF60A, SS18 and BAF47) and full-length FET proteins (EWSR1 and FUS) remaining on sepharose-bound FUS-NTD‑GST pulldown baits after increasing stringency washes with 100, 150, 250, 500 or 1000 mM NaCl. Note loss of BRG1 at NaCl concentration > 250mM
Figure 3A. Immunoblot analysis (IB) of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from MLS cell line 402‑91 and EWS cell line TC-71. Antibodies against BRG1, DDIT3 (FUS-DDIT3), the C-terminal parts of normal FUS and EWSR1, and FLI1 (EWSR1-FLI1) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. B. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for FUS‑DDIT3, EWSR1 and FUS in MLS 402-91 cell line and EWSR1-FLI1, EWSR1 and FUS in EWS TC-71 cell line. Mean ± SEM is shown with individual replicates indicated by circles (n=3). Student's t-test, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data. C. Immunoblot analysis of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED, EWSR1-FLI1-EGFP or dsRED are shown as representative immunoblots. Antibodies against BRG1, DDIT3 (FUS-DDIT3-dsRED), the C‑terminal parts of normal FUS and EWSR1, EZH2 and FLI1 (EWSR1-FLI1-EGFP) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. Immunoblots from all replicates are shown in the source data (with either dsRED- or EGFP‑tagged proteins expressed).D. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for the fusion protein (FUS‑DDIT3 or EWSR1-FLI1), EWSR1, FUS and EZH2 in the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED/-EGFP (n=4, circles), EWSR1-FLI1-EGFP (n=3, squares) or dsRED/EGFP (n=4, triangles). Mean ± SEM is shown with individual replicates indicated. Student's t-test, no significant changes, p>0.05. Original data for all quantifications, including p-values, are shown as source data.
Figure 4A. Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‑DDIT3‑EGFP or EWSR1‑FLI1‑EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection and by squares from two experiments with transient transfection (24h and 48h , n=4. Student's t-test, ns= not significant, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.
Figure 1 (A, B) t-SNE visualization of 20,009 human retinal cells colored by (A) annotation of 18 transcriptionally distinct clusters (C0-C17) and (B) their distribution in 3 donor retina samples.   C) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥10 nTrans (number of transcripts).  D) t-SNE plots showing expression of a set of selected marker genes for major retinal classes. E) Correlation matrix for the identified 18 clusters. The upper triangle depicts the z-value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters.  F) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered by cluster (C0-C17).
Figure 2 A) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters. Brown colour indicates ≥100 nTrans (number of transcripts).  B) Representation of the three donor retina samples in the six rod photoreceptor clusters.  C) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters.  D) Distribution of rod photoreceptor populations with high MALAT1 expression (MALAT1-hi) or low MALAT1 expression (MALAT1-lo) in three donor retina samples.  E) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer (ONL). INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm.
Figure 3 A, B) (A) t-SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters (CCA0-CCA12) and (B) their distribution in 3 donor retina samples.  C) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥50 nTrans (number of transcripts).  D) t-SNE plots showing expression of MALAT1.  E) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1-hi (CCA1, CCA10) and MALAT1-lo (CCA0) subpopulations. x-axis depicts normalized transcript levels.  F) t-SNE plots showing expression of major ribosomal genes.  G) Heatmap of differentially expressed genes between the two MALAT1-hi clusters CCA1 and CCA10. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered in CCA1 and CCA10.
Figure 4 Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post-mortem (7 and 13 hours, Retina 7), showing decreases in MALAT1-hi rod subpopulations in the outer nuclear layer (ONL) at later time point. INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm. White arrows indicated MALAT1-hi rod photoreceptors.
Figure 5 A) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters. Brown colour indicates ≥10 nTrans (number of transcripts).  C) Scatter plots showing expression of OPN1LW/OPN1MW or OPN1SW in individual cone photoreceptors for population C10. The colour depicts expression level for OPN1LW/OPN1MW in individual cells.  D) Heatmap of top 20 differentially expressed genes between L/M cones and S cones. The colour depicts normalised gene expression (z-score capped at 2.5).  E) Expression pattern of glial markers in Muller glia (C9) and retinal astrocytes (C16). x-axis depicts normalized transcript levels.
Figure 6C) t-SNE analysis of the human Müller glia cell line MIO-M1 with the retinal cell types identified in this human neural retina atlas.
Figure 1B. Southern blotting confirms successful targeting. Introduction of an additional EcoRI site results in a 4.1 kb restriction fragment detectable by Southern for targeted ES cells (A174T/+) but not for parental DNA (+/+).C. Capillary sequencing for Rnaseh2b+/+, Rnaseh2bA174T/+, and Rnaseh2bA174T/A174T DNA confirmed the presence of the introduced missense mutation.D. Mouse genotyping by multiplex PCR. Top: A 221 bp PCR product is present in wild-type mice (+/+); the Rnaseh2bA174T allele (also) give a 460 bp product. Bottom: Position of forward (x) and reverse primers (y, z).E. Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T/A174T MEFs and RNASEH2BA177T/A177T LCLs. Representative of three independent experiments.G, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.
Figure 3A. Validation of Rnaseh2b-/- MEF lines. RER activity (DRD:DNA) is undetectable in a Rnaseh2b-/- line consistent with complete inactivation of the Rnaseh2b gene (Reijns et al, 2012). Mean of three independent experiments; error bars, SEM, **** = p<0.0001, two-tailed t-test. Rnaseh2b-/- and Rnaseh2b+/+ control MEFs on a C57BL/6 p53-/- background.B. ISG transcript levels are increased in Rnaseh2b-/- MEFs.C. BeadArray transcript analysis (Illumina) detected induction of multiple ISGs, including the cytokines CXCL10 and CCL5, but not other cytokine transcripts in Rnaseh2b-/- MEFs. Plotted, average fold enrichment versus p-value of significantly upregulated transcripts (p<0.05, after multiple testing correction) comparing two Rnaseh2b-/- MEF lines versus 4 Rnaseh2b+/+ MEF lines. 17 out of 29 transcripts are ISGs.D. CXCL10 and CCL5 transcripts (detected by RT-qPCR) are significantly elevated.E. Increased CXCL10 and CCL5 protein (detected by ELISA) is secreted by Rnaseh2b-/- MEFs. (B, D, E) Mean data from three experiments for six independent Rnaseh2b-/- MEF lines versus four independent Rnaseh2b+/+ MEF lines. Error bars, SEM. * = p<0.05, ** = p<0.01, Mann-Whitney U test.
Figure 4A. ISG activation and cytokine secretion in Rnaseh2b-/- MEFs is markedly impaired by cGAS or STING siRNA depletion. Upper left and lower panels: RT-qPCR of IFIT1, cGAS and STING transcripts, 48 h after siRNA targeting luciferase (control), cGAS or STING. Upper right panel: CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion. Concentration of CCL5 (ELISA), normalised to luciferase siRNA control levels in each experiment. Mean from three independent experiments using one Rnaseh2b-/- MEF line; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test for RT-qPCR, one sample t-test for CCL5ELISA.B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test.B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test.E. ISG induction in Rnaseh2bA174T/A174T mice is STING dependent. RT-qPCR of RNA extracted from hearts from Sting+/+ Rnaseh2bA174T/A174T (n=4) and Sting-/-Rnaseh2bA174T/A174T (n=6) three-month old mice.F. Absence of STING does not significantly decrease basal ISG expression. RT-qPCR of RNA extracted from hearts from Sting+/+ (n=3) and Sting-/- (n=5) three-month old mice. (E, F) Each data point represents the mean of technical triplicates from one mouse. Error bars, SEM. * = p<0.05, Mann-Whitney U test.
Figure 5A. Overexpression of RNase H1 in Rnaseh2b-/- cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wildtype levels, while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b-/- MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n=3 independent experiments ± SEM.B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test.B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test.D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.
Figure 1(b) Chromatin immunoprecipitation using α-FLAG antibody was performed to confirm the pull-down of Sα chromatin from the Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The a, b, and c bars indicate the primers used for ChIP-qPCR as shown in Fig. 1a (n=3; mean + sd; two tailed unpaired Student's t-test).(e) Bar graph showing the proteins with ≥2-fold enrichment as calculated by the heavy to light SILAC ratio.(f) Effect of the siRNA-mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3-2A cells. Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=2; mean + sd).
Figure 2(a) Top: Scheme of the IgA-switching assay in CH12F3-2A cells. After electroporation of siRNAs, cells were cultured for 24 h, and then stimulated by CIT, cultured for another 24 h, and subjected to FACS analysis. Bottom: Confirmation of the siRNA-mediated KD of SAMHD1 in CH12F3-2A cells.(b) FACS profile of the percentage of CH12F3-2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3-2A cells cultured with (+) or without (-) CIT stimulation for 24 h. The number in the FACS plot represents the percentage of cells expressing IgA on their surface.(c) Summary of the FACS data obtained from three independent experiments. Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=3; mean + sd; two tailed unpaired Student's t-test).(d) Quantitative RT-PCR analysis of the μGLT, αGLT, Aid, and Samhd1 expression in the indicated samples. The data were normalized to Gapdh, and represents as the mean of the two independent experiments with standard deviations. Statistical significance as determined by two-tailed unpaired Student's t-test is shown. ns = not significant.(e) Confirmation of the CRISPR/Cas9 mediated KO of Samhd1 in CH12F3-2A cells. Beta-actin was used as a loading control.(f) CSR time course analysis of WT and Samhd1 KO clones. The data are shown as percentage of IgA expressing cells (n=2; mean ± sd; two tailed unpaired Student's t-test; **, p<0.01).(g) Proliferation of WT and Samhd1 KO clones after stimulation with CIT (n = 2; mean ± sd; two tailed unpaired Student's t-test; ns = not significant).(h) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3-2A. Histograms show the CFSE dye dilution derived from the indicated samples. Aphidicolin (2 μg/mL) was also used as a positive control.(i) Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells. Bottom: Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice. Tubulin was used as a loading control.(j) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments. The data are shown as percentage of IgG1 (left) or IgG3 (right) expressing cells (n=5; mean ± sd; two-tailed unpaired Student's t-test).
Figure 3(b) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. CH12F3-2A cells were transfected with the indicated siRNAs. (c) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3-2A cells by SAMHD1 siRNA or by tetracycline (50 nM) treatment, respectively. Expression of Tubulin is shown as a loading control (e: left). Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig. 3e (e: right).Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments (f). Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test) (g).(h-j) Analysis of the IgH/c-Myc translocation frequency in WT and Samhd1 KO CH12F3-2A cells. (h) Schematic illustration of the experimental design for the translocation assay. (i) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (j) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).(k-m) Analysis of the IgH/c-Myc translocation frequency in spleen B cell derived from WT (Samhd1+/+) or Samhd1 KO (Samhd1-/-) mice. (k) Schematic illustration of the experimental design for the translocation assay. (l) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (m) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).
Figure 4(b) Mutation frequency in the 5′ Sµ region of WT or Samhd1 KO CH12F3-2A cells. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.(d) LM-PCR analysis of the AID-induced DNA double-strand breaks in the Sµ region of WT or Samhd1 KO CH12F3-2A cells. Wedges indicate a three-fold increase in the DNA amount. Amplification of Gapdh was used as an internal loading control.(e) Gamma-H2AX-ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA (% IP) normalized to the total amount of DNA used for the immunoprecipitation (n=3; mean + sd; two-tailed unpaired Student's t-test; ns = not significant).(f) Left: Scheme of long-range interactions between Sμ-Sα elements in the IgH locus before and after AID activation. Right: Representative gel picture of the 3C assay detecting the Sμ-Sα interaction in WT or Samhd1 KO CH12F3-2A cells with or without CIT stimulation for 24 h. Gapdh was amplified as a loading control.
Figure 5(a-d) Analysis of Sμ-Sα recombination junctions from WT or CH12F3-2A cells after transfection with control (siCONT) or Ligase 4 (siLIG4) siRNA. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to junction analysis. A total of 62 (siCONT) and 73 (siLIG4) junctions from WT, and 60 (siCONT) and 60 (siLIG4) junctions from Samhd1 KO cells were sequenced and analyzed. Statistical significance as evaluated by Fisher's exact test (b) or two-tailed unpaired Student's t-test (d) or is shown. Schematic representation of the Sμ-Sα junction analysis (a). Summary of the characteristics of Sμ-Sα recombination junctions (b). Sequences at Sμ-Sα recombination junctions with insertions (c). Summary of the average length of insertions at Sμ-Sα recombination junctions (d).(e-i) Analysis of the CRISPR/Cas9 induced chromosomal translocations between the Rosa26 (Chr6) and H3f3b (Chr 11) genes. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to chromosomal translocation or junction analysis. Schematic representation of the chromosomal translocations (e). Representative images of agarose gels after PCR-amplification of the Rosa26/H3f3b translocation from WT or Samhd1 KO CH12F3-2A cells (f). Relative frequency of Rosa26/H3b3f chromosomal translocations (g). Frequency of junctions containing insertions (h) and average length of insertions (i). Statistical significance as evaluated by two-tailed unpaired Student's t-test (g, i) or Fisher's exact test (h) is shown.