buffer b  (Fisher Scientific)

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Purification, Mobility Shift

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1 promotes BLM-mediated unwinding of telomere R-loops. ( A ) (Top) Schematic of the oligo-based telomere R-loop unwinding assay. Telomere R-loop substrates were generated by hybridizing 32 P-labeled TERRA and two telomere DNA fragments (TDR2 and TDR3). TRF2 and/or RAP1 were pre-incubated with the R-loops and then BLM was added to the reaction, and the complex was resolved by 10% native polyacrylamide gel electrophoresis to monitor for R-loop unwinding. Displacement of the invading radiolabeled TERRA from R-loops indicates that R-loop unwinding. (Bottom) The TRF2–RAP1 complex promotes BLM-mediated unwinding of telomere R-loops. The effects of TRF2 alone (40, 80 nM) or in combination with RAP1 (20, 40, 80 nM) on the ability of BLM (20 nM) to unwind telomere R-loops were examined. 32 P-labeled TERRA and R-loops were resolved by native-PAGE and shown in lanes 1 and 2. ( B ) Quantification of BLM-mediated R-loop unwinding reactions in panel (A). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .9485); **** P < .0001. ( C ) The TRF2 basic domain is required for efficient unwinding of telomere R-loops. The effect of WT TRF2, TRF2 ΔB , TRF2 ΔB,L288R , and RAP1 to enhance BLM-mediated telomere R-loop unwinding was examined. The sizes of 32 P-labeled TERRA and R-loops were resolved by native-PAGE, as shown in lanes 1 and 2. ( D ) Quantification of BLM-mediated R-loop unwinding reactions in panel (C). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. **** P < .0001. ( E ) The TRF2–BLM interaction enhances telomere R-loop unwinding. The effect of TRF2–RAP1 on the ability of WT and mutant BLM (3A or P690L) to unwind telomere R-loops was tested as in Fig. . 32 P-labeled TERRA and R-loops were loaded as size markers (lanes 1 and 2) and resolved by native-PAGE. ( F ) Quantification of the percentages of unwound R-loops in panel (E) as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .98); **** P < .0001.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Generated, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Clear Native PAGE, Mutagenesis

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: BLM preferentially releases TERRA over ssDNA from telomere D/R-loops. ( A ) Schematic of the assay used to measure how TRF2–RAP1 promotes BLM-mediated unwinding of RAD51/ssDNA and RAD51AP1/TERRA-generated telomeric D/R-loops. Telomere D/R-loops were generated by incubating RAD51 with IRDye-700-labeled telomere ssDNA (red), RAD51AP1 with IRDye-800-labeled TERRA (green), and telomere plasmids together as described in Fig. . Native plasmid-sized telomere D/R-loops were obtained after deproteinization and column purification. BLM with or without TRF2–RAP1 was then incubated with these D/R-loops, and ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. ( B ) BLM preferentially releases TERRA over ssDNA from telomere D/R-loop. BLM (20, 40, 80 nM) was tested for its ability to unwind telomere D/R-loops or TRF2–RAP1-bound D/R-loops. ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. The unwinding of telomere D/R-loops by BLM was enhanced by TRF2–RAP1. ( C ) Quantification of the amount of D- and R-loops relative to the negative control (no proteins, lane 1). Data were plotted as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. * P = .02282; ** P = .001278; *** P = .0007284; **** P < .0001. ( D ) The effects of TRF2–RAP1 on WT BLM, the helicase-dead BLM K695R or BLM mutants on D/R-loop unwinding were tested as in panel (B). In contrast to WT BLM, TRF2–RAP1 cannot enhance BLM ’s ability to unwind telomere D/R-loops. D/R-loop unwinding was analyzed by 1% agarose gels. ( E ) Quantification of the relative amounts of D-loops or R-loops to the control without proteins (lane 1) is shown as mean ± SD from three independent experiments. ANOVA test was used to evaluate statistical differences. ns: non-significant ( P = .15; .4147; .8026); ** P = .001193; *** P = .000158.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Generated, Labeling, Plasmid Preparation, Purification, Incubation, Negative Control, Control

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1–BLM is required to resolve telomere R-loops in U2OS cells. ( A ) U2OS cells expressing TRF2 ΔB, L288R were treated with shControl, shBLM, or shTRF2. Immunofluorescence-FISH analysis of cells containing UTs (PNA telomere probe, red) co-localized with R-loops (S9.6 antibody, green) and DAPI-stained nuclei (blue). White arrow: co-localization of R-loops on UTs. U2OS cells expressing shBLM-resistant WT BLM cDNA and indicated BLM mutants were treated with shBLM, shTRF2, and TRF2 ΔB, L288R . IF-FISH analysis was performed to detect UT/R-loop co-localization. White arrow: co-localization of R-loops on UTs. ( C ) Quantification of data from Fig. and , showing the number of UT/R-loop colocalizations per U2OS cell. Data from three independent experiments is shown as mean ± SEM from minimum 200 nuclei per experiment. Statistical evaluation was performed by one-way ANOVA test. ns: non-significant ( P > .9999); ** P = .0032; .0035; .0062; .0092; .0052; **** P < .0001. ( D ) Model showing that TRF2–RAP1 inhibits telomere HDR by promoting BLM-mediated telomere R-loop removal. RAD51AP1 and TERRA-dependent R-loops promote RAD51-mediated telomere D-loop formation. The TRF2–RAP1 complex and TRF2–BLM interaction are required to promote BLM helicase-mediated unwinding of telomere R-loops and then D-loops. The RAP1–TRF2–BLM complex represses HDR on telomeres by removing R-loops to inhibit telomere D-loop formation.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , Schematic for snATAC–seq and nanoCUT&Tag experiments in adult human CNS tissue. b , Two-dimensional UMAP of the snATAC dataset colored by clusters and labeled by cell type. c , Gene activity scores for different genes in the identified cell types. d , Two-dimensional UMAP of the H3K27ac nanoCUT&Tag dataset with cell annotations based on integration with snATAC–seq. e , Heatmap showing snATAC–seq differentially accessible peaks across different clusters and cell types. f , nanoCUT&Tag genome browser snapshot showing H3K27ac (top) and H3K27me3 (bottom) pseudobulk signal distribution across different marker genes for each cell type. g , nanoCUT&Tag meta-signal enrichment plots for H3K27ac (green) and H3K27me3 (red) in the MOL population. Top: line plots showing signal enrichment for the two modalities at different peak sets. Middle and bottom: heatmap showing H3K27ac (middle) and H3K27me3 (bottom) signal enrichment across different peak sets. Peak sets (left to right)—H3K27me3 peaks, H3K27ac peaks, ATAC peaks and ATAC peaks from a previously published dataset . h , Trimodal clustering of the genome highlights patterns of signal distribution across all cell types. i , Correlation matrix of ATAC, H3K27ac and H3K27me3 signals in each cell type shows strong correlation between active marks for individual cell types, and anticorrelation with the repressive H3K27me3. UMAP, uniform manifold approximation and projection; norm., normalized. Schematic in a created in BioRender; Castelo-Branco, G. https://biorender.com/orfbpyi (2025).

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques: Labeling, Activity Assay, Marker

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , Cumulative distribution of the co-accessibility score for all loops identified by Cicero. Red line shows the score cutoff (0.5) used for assessing high-quality interactions and captures the top 5% of all loops. b , snATAC-seq genome browser snapshot showing chromatin accessibility signal in all cell types at CDC42EP1 locus (left), identified SOX10-distal enhancer (middle) and SOX10 locus (right) and the corresponding loops identified using Cicero. Red columns highlight the CDC42EP1 and SOX10 genes, and the gray column highlights the new enhancer. c , UCSC Genome Browser snapshot showing the identified enhancer locus (light blue column) as well as overlap with previously identified ENCODE cCREs, PhyloP base conservation score and evolutionary conservation with different species. d , snATAC-seq genome browser track showing chromatin accessibility in OPCs and MOLs at the newly identified enhancer, SOX10 locus, and previously characterized U1-U5, D6 and D7 enhancers (purple) and overlap with thyroid hormone receptor motifs (T3R, green) and TFAP2A motifs (orange). e , snATAC-seq and nanoCUT&Tag genome browser tracks the CDC42EP1-enhancer-SOX10 locus showing ATAC, H3K27ac and H3K27me3 pseudobulk signal in MOLs and OPCs and Cicero links. f , FACS plots showing transduction efficiency (left) and gene expression changes (qPCR) of SOX10 when directing dCas9–p300 to the U2 enhancer, U3 enhancer, and a nontargeting control (NTC) (right). N = 4 biological replicates; data shown as mean ± s.e.m. Statistics: one-way ANOVA with Tukey’s post hoc test. Two-sided t-test performed. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, NS = nonsignificant. g , Scatterplot showing TF strength of shared core TFs in MOLs (x axis) and OPCs (y axis) and highlighting the identified HOX genes. h , List of identified HOX genes being differentially accessible in spinal cord OPCs and MOLs. i , Scatterplot showing correlation between the multiome-RNA and multiome-ATAC. j , Stacked violin plot showing gene expression levels of all HOX genes in OPCs in all regions from an adult human brain transcriptomic atlas .

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques: Transduction, Gene Expression, Control

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , Volcano plot showing differentially accessible peaks in SC and cortical OLGs. Previously characterized marker genes are shown in red and labeled. HOX cluster-associated peaks are shown in blue. Two-sided t test with Benjamini–Hochberg correction. Thresholds, adjusted P = 0.001, log(FC) = 1.5. b , Same as in a , but highlighting the specific HOX clusters identified as being differentially accessible. Two-sided t test with Benjamini–Hochberg correction. c , snATAC–seq genome browser snapshot showing pseudobulk chromatin accessibility signal in AST, MIGL, MOL and OPC populations from the CSC and motor cortex at the HOXA locus. MIGL signal is depleted in both regions, whereas AST, OPC and MOL exhibit accessibility in SC. d , Schematic showing workflow for the multiome experiment and UMAP embedding of the multiome ATAC with cell types annotated using the multiome-RNA. e , Stacked violin plots showing the expression (top) and promoter accessibility (bottom) in cortex and SC-derived MOLs and OPCs from a multiome experiment. Most differentially accessible HOX genes and OLG marker genes are shown. Schematic in d created in BioRender; Castelo-Branco, G. https://biorender.com/orfbpyi (2025).

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques: Marker, Labeling, Expressing, Derivative Assay

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , nanoCUT&Tag and snATAC–seq genome browser tracks showing H3K27ac, H3K27me3 and ATAC pseudobulk signals in OLGs at the HOXA in CSC (upright track, darker shade) and motor cortex (inverted track, lighter shade). b , Same as in a , but for the HOXD locus. c , Gaussian smoothed normalized signal from ATAC (blue), H3K27ac (green) and H3K27me3 (red) across the HOXA cluster with a 50-kb flanking region upstream and downstream. Gray bars show the locations of the cumulative ‘signal boundaries’ identified in each modality. Color intensity reflects the cumulative strength of the signal boundary. d , Same as in c , but with each modality separated out. HOXA directionality is shown at the top, and arrows beneath show the medium (two modalities) and strong (three modalities) signal boundaries. e , nanoCUT&Tag and snATAC–seq genome browser track of the HOXA cluster showing the location of the strong signal boundaries and the corresponding inactive, primed and silenced chromatin domains. f , nanoCUT&Tag and snATAC–seq genome browser tracks showing the ATAC (blue), H3K27ac (green) and H3K27me3 (red) pseudobulk signal in the microglial and astrocyte populations at HOXD in both SC (CSC) and cortex (BA4). g , nanoCUT&Tag genome browser track around the HOXD locus (marked with dotted lines) with H3K27me3 (red) and H3K27ac (green) pseudobulk signal in SC OLGs. Single-cell tracks are shown below and sorted by decreasing H3K27me3 signal. Group 1 cells exhibit moderate H3K27me3 at the 3′ end, while group 2 cells show H3K27me3 depletion, and the amount of H3K27ac remains the same in both groups, suggesting that group 2 cells may be expressing low levels of HOX genes.

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques: Single Cell, Expressing

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , snATAC-seq and nanoCUT&Tag genome browser tracks showing H3K27ac, H3K27me3 and ATAC pseudobulk signal in OLGs at the HOX-B and HOX-C clusters in cervical spinal cord (upright track, darker shade) and motor cortex (inverted track, lighter shade). Directionality of the clusters is shown by the arrow. b , Gaussian smoothed normalized signal from ATAC (blue), H3K27ac (green) and H3K27me3 (red) in spinal cord OLGs (solid line) and cortical OLGs (dotted line) across each HOX cluster with a 50 kb flanking region upstream and downstream. c , Gaussian smoothed normalized signal from ATAC (blue), H3K27ac (green) and H3K27me3 (red) across the HOXB, HOXC and HOXD clusters with a 50 kb flanking region upstream and downstream, separated by each modality. Gray bars show the location of ‘signal boundaries’ identified in each modality. Dotted lines mark the boundaries of each HOX cluster. Arrowheads underneath the plots mark intermediate and strong signal boundaries. HOX cluster directionality shown by arrow on top. d , snATAC-seq and nanoCUT&Tag genome browser tracks showing the ATAC (blue), H3K27ac (green), and H3K27me3 (red) pseudobulk signal in the microglial and astrocyte populations at HOXA in both spinal cord (CSC) and cortex (BA4).

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques:

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , Normalized Micro-C contact matrix at 5-kb resolution at HOXA (left) and HOXD (right) loci and corresponding ATAC, H3K27ac and H3K27me3 tracks in human adult SC OLGs showing the c-Dom and t-Dom TAD structures, including the sub-TAD contacts. Contacts with distal enhancers is shown by the gray bars. b , Micro-C contact matrix showing contacts between HOX genes and flanking enhancers in hOPCs, in contrast to B cells. c , Correlation matrix of ATAC, H3K27ac and H3K27me3 signals in cortex and SC-derived OLGs across all four HOX clusters. d , Contact matrix showing long-range interaction between miR10b and LINC01116, virtual 4c (anchored on LINC01116) H3K27me3, H3K27ac, ATAC and inferred loops are shown. e , scATAC, H3K27ac and H3K27me3 tracks showing signal distribution at HOXD and distal LINC01116 in SC OLGs and cortex OLGs. f , Schematic showing plasmid and lentivirus setup for CRISPRi/a experiment. g , Gene expression changes (qPCR) of five genes from the CRISPRi/a experiment. Left, data from CRISPRa (left, dCas9–p300) and CRISPRi (right, dCas9–KRAB) targeting either miR10b (turquoise), LINC01116 (red) or an NTC (gray). n = 4 biological replicates; data shown as mean ± s.e.m.; statistics, one-way ANOVA with Tukey’s post hoc test; two-sided t test performed; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ANOVA, analysis of variance. Schematic in f created in BioRender; Castelo-Branco, G. https://biorender.com/orfbpyi (2025).

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques: Derivative Assay, Plasmid Preparation, Gene Expression

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , Schematic of the adult human brain showing the location of pontine and thalamic gliomas along the A-P axis. Schematic in a created in BioRender. Castelo-branco, G. https://BioRender.com/orfbpyi (2025). b , snATAC-seq and nanoCUT&Tag normalized promoter accessibility (ATAC), H3K27ac and H3K27me3 signal in spinal cord OLGs (top) and cortical OLGs (bottom) at different developmental genes associated with brain and spinal cord patterning. c , Genome browser tracks showing nanoCUT&Tag H3K27ac and H3K27me3 pseudobulk signal coverage at the HOXA cluster in spinal cord (SC) derived adult human OLG (hOLG) and PFA-EP tumors, H3.3K27M pontine tumors, and H3.3K27M thalamic tumors . d , Same as c , but at the HOXD locus.

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques: Derivative Assay

Journal: Nature Neuroscience

Article Title: Single-nucleus epigenomic profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

doi: 10.1038/s41593-026-02208-0

Figure Lengend Snippet: a , nanoCUT&Tag and snATAC–seq normalized promoter accessibility (ATAC), H3K27ac and H3K27me3 signal in SC OLGs (top) and cortical OLGs (bottom) at all HOX genes. Asterisk indicates the genes previously identified to be expressed in pontine HGGs . b , Normalized Hi–C contact matrix in H3.3K27M pontine HGG at the HOXA locus (marked by dotted lines) and corresponding ATAC, H3K27ac and H3K27me3 signals in SC OLGs and H3K27ac and H3K27me3 in H3.3K27M pontine HGG, showing similarity in mark distribution in nondiseased conditions and gliomas. c , Aggregate pileup analysis of hOPC loops (Micro-C) at HOXA (top) and HOXD (bottom) in pontine HGG, PFA-EP and thalamic HGG (left to right) . d , Insulation score from the Micro-C matrix across a 5-Mb window spanning HOXA (left) and HOXD (right) in the three HGGs (green, pink, yellow, from ref. ) and B cells (blue) overlaid on the hOPC insulation profile (black).

Article Snippet: Nuclei of 8 μl were added to 7 μl ATAC buffer B (10x Genomics) and loaded onto the Chromium Chip H. GEM incubation and post-GEM incubation clean-up were performed according to Chromium Next GEM Single Cell ATAC Reagent Kits (v1.1) instructions (step 2.0–3.2).

Techniques: Hi-C, Insulation