rabbit polyclonal anti chd8  (Novus Biologicals)

Journal: iScience

Article Title: A CHD8-TRRAP axis facilitates MYC and E2F target gene regulation in human neural stem cells

doi: 10.1016/j.isci.2025.111978

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal Anti-CHD8 , Bethyl , Cat# A301-224A; RRID: AB_890578.

Techniques: Virus, Recombinant, Membrane, Protease Inhibitor, SYBR Green Assay, Staining, cDNA Synthesis, Mass Spectrometry, shRNA, Sequencing, Control, Plasmid Preparation, Software, Sonication

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Isoforms and functional domains of the chromodomain-helicase-DNA-binding protein 8 (CHD8) protein. The three isoforms of CHD8 protein, including (1) CHD8-S, a short isoform; (2) CHD8-L1, a long isoform; and (3) CHD8-L2, a long isoform . CHD8-L1 and CHD8-L2 are composed of two histone-binding chromodomains (C1 and C2, yellow), a chromatin-remodeling helicase domain (helicase, cyan), multiple protein-interacting chromatin organization modifier domains (CR, magenta), and a DNA-binding brahma and kismet domain (BRK, pink) . The position of the identified variant relative to CHD8-L1 and CHD8-L2 isoforms is indicated in red.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Functional Assay, Binding Assay, Variant Assay

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Variant validation using Sanger sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR). (a) The Sanger chromatograms indicate the absence of the variant in the maternal DNA and presence in the proband fibroblasts and blood DNA, indicating a nonmaternal inheritance of the variant. (b) Two sets of cDNA primers, including a set of primers upstream of the variant and another downstream of the variant (Table and Figure ), were used to conduct qRT-PCR on CHD8 cDNA in the proband line versus the control lines. (c) CHD8 transcripts captured by both upstream and downstream cDNA primers showed significant reduction (upstream primers: ~42%, downstream primers: ~33%) in the CHX− proband samples relative to that of controls (Wilcoxon test: p = 0.0313 for both primer sets). CHX+ samples of both the proband and the controls showed equivalent levels of CHD8 transcripts.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Variant Assay, Biomarker Discovery, Sequencing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Immunoblotting and mass spectrometry–based proteomic analysis. (a) Western blots indicating the level of CHD8 protein detected from controls (C1, C2) and proband (P) samples. Three technical repeats ( n = 3) of Western blotting using the CHD8 C-terminal antibody (Cell Signaling Technologies #11891, 1:1000) showed the relative quantities of CHD8-L1 and CHD8-L2 against GAPDH (loading control). (b) Protein band quantification of the Western blots showed a significant reduction of the CHD8-L1 and CHD8-L2 isoform levels in the proband (P) (L1: ~51%, L2: ~48%) compared to those of the controls (C) (Mann–Whitney test: p = 0.0089, p = 0.0238, respectively). (c) The abundance of CHD8 is ranked significantly lower in the proteome of the proband compared to the controls. (d) The abundance of CHD8 is significantly lower in proband fibroblasts (70%, red dot) and lies outside of the control range (80%–104%, n = 5). (e) Volcano plot showed the relative amount of proteins in the proband line compared to the controls, with vertical lines indicating +/−1.5 log 2 -fold change and the horizontal line indicating statistical significance. CHD8 is reduced significantly by ~30% ( p < 0.001) in the proband line compared to the controls. MeCP2 (green) is significantly reduced by ~43% ( p < 0.01), whereas bromodomain adjacent to zinc finger domain 1A ( BAZ1A ) encoding the accessory subunit of the ATP-dependent chromatin assembly factor (ACF) (orange) is significantly increased by ~72% ( p < 0.001). CHD8-regulated proteins (purple), including acylglycerol kinase (AGK), CDC42-binding protein kinase (CDC42BPB), phosphatase and tensin homolog (PTEN), and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), showed a reduction in their corresponding protein abundance, with AGK being the highest at ~55% ( p < 0.001). Transportin 3 (TNPO3), nuclear receptor corepressor 1 (NCOR1), and proteasome assembly chaperone 2 (PSMG2) showed an increase of abundance with TNPO3 being the highest at ~39% ( p < 0.001). (f) STRING network analysis revealed coexpression (black), interactions (magenta), and comentions in literature (lime green) between CHD8, MeCP2, CDKL5, FOXG1, and ACF.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Western Blot, Mass Spectrometry, Control, MANN-WHITNEY, Binding Assay, Phospho-proteomics, Quantitative Proteomics

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Genetic strategy targeting exon 4 of the Chd8 gene. The exon was flanked with loxP sites to excise the loci, resulting in a frameshift mutation that disrupts the production of the CHD8 protein after Cre excision. (B and C) Representative images of CHD8 in the CA1 regions of adult mouse brains. CHD8 is expressed in neurons and astrocytes in controls (B). After tamoxifen administration, Chd8 fx/fx : CAGGS-CreER +/ − mouse brains showed non-detectable levels of CHD8 protein (C). In both (B) and (C), arrows point to astrocytes expressing CHD8 and its knockout thereafter. (D) Schematic diagram for tamoxifen-induced Chd8 cKO, the stab-wound injury model, and the analysis of reactive gliosis. (E and F) Response of GFAP + astrocytes and Iba1 + microglia after stab-wound injury. Control mice (E) exhibit astrocytic and microglial response as expected, while global Chd8 cKO mice (F) show reduced staining for both GFAP + and Iba1 + along the needle track. (G and H) High-magnification images from the injury site from the corresponding genotypes. Note the reduction in cell body size, process elongation, and polarity in astrocytes from global Chd8 cKO mice (H). (I and J) Decreased area occupied by GFAP + astrocytes (I) and Iba1 + microglia (J) in global Chd8 cKO mice compared to controls. In (B) and (C), scale bars indicate 20 μm; in (G) and (H), scale bars indicate 50 μm; in (E) and (F), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified. In (E)–(H), the dashed lines indicate the needle track of the injury. In (I) and (J), data points illustrate the quantified area from the six brain slices most proximal to the injury epicenter. Data are normalized to the means of the ipsilateral site in control mice. **** p < 0.0001, ns, not significant; statistical analysis was performed with two-way ANOVA; on the violin plots, dashed lines indicate the 25%, mean, and 75% percentile, respectively, from bottom to top; n = 4 mice per genotype.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: Mutagenesis, Expressing, Knock-Out, Control, Staining

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Strategy for tamoxifen-induced, astrocyte-specific Chd8 cKO utilizing the Aldh1l1-CreERT2 line. Mice were crossed with the Ai14- tdTomato reporter line to visualize recombined cells. (B and C) Astrocyte-specific Chd8- cKO mice show non-detectable CHD8 protein expression in cortical slices. Controls are mice without the Chd8 floxed alleles but expressing Aldh1l1-CreERT2 to turn on the expression of tdTomato reporter. Arrows indicate CHD8 expression in tdTomato + astrocytes in control but not astrocyte Chd8 cKO. (D and E) GFAP and Iba1 staining of astrocytes and microglia, respectively, after stab-wound injury in control (D) and astrocyte cKO mice (E). (F and G) High-magnification images from the injury sites from the corresponding genotypes showing reduction in cell body size, process elongation, and polarity in astrocytes and reduced microglia numbers in astrocyte cKO mice. (H and I) Decreased area occupied by GFAP + astrocytes and their numbers in astrocyte cKO mice compared to controls. (J and K) Decreased area occupied by Iba1 + microglia and their numbers in astrocyte cKO mice compared to controls. In (H)–(K), data points represent the quantified area from the six brain slices most proximal to the injury epicenter. Data are normalized to the means of the ipsilateral site in control mice. In (B) and (C), scale bars indicate 20 μm; in (F) and (G), scale bars indicate 50 μm; in (D) and (E), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified. In (D)–(G), the dashed lines in merged images indicate the needle track of the injury. In (H)–(K), *** p < 0.001; **** p < 0.0001; ns, not significant; statistical comparisons were analyzed with two-way ANOVA; on the violin plots, dashed lines indicate the 25%, mean, and 75% percentile, respectively, from bottom to top; n = 6 mice per genotype.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: Expressing, Control, Staining

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Heatmap of DEGs identified through RNA-seq between control and astrocyte Chd8 cKO mice after LPS treatment ( n = 5 mice per genotype). A total of 109 DEGs were identified, 76 of which were upregulated and 33 were downregulated (FDR < 0.05). (B) Volcano plot depicting the distribution of upregulated and downregulated genes, relative to their quantified fold change and their corresponding p values. The threshold was set at p (adjusted) < 0.05. (C) Venn diagram of the detected DEGs, depicting a subset of DEGs that correspond to genes whose expression is specific to astrocytes, neurons, and microglia. Of those, many DEGs (41) were determined to be astrocyte specific, while fewer were deemed to be specific in neurons (4) and microglia (1). (D) Bar plot showing the fold enrichment of the detected DEGs in our dataset, indicating significant enrichment of DEGs for astrocyte marker genes. Notably, no enrichment was detected when comparing neuronal or microglial genes to the cell-type markers from previous studies, as cited. *** p < 0.001; **** p < 0.0001. (E) GO terms analysis reveals changes associated with many cellular processes, including lipid and metabolic pathways in astrocyte cKO mice in response to LPS stimulation. (F) Heatmap of the top 20 DEGs identified through RNA-seq. Of these, 13 were upregulated and 7 were downregulated in astrocyte cKO mice treated with LPS vs. control mice treated with LPS. (G–O) qPCR analysis of Gstt3 (G), Acsl3 (H), Etnppl (I), Phykpl (J), Gjb6 (K), Slc9a8 (L), Agt (M), Tnfrsf25 (N), and Lcat (O) mRNA confirms the altered expression shown in (F) ( n = 4 mice per group). (P) Representative western blotting of TNFRSF25 from cortices of control and astrocyte cKO mice after LPS administration, showing reduced TNFRSF25 protein in the cortex of astrocyte cKO mice ( n = 7 mice per group). (Q) Representative western blotting of LCAT from cortices of control and astrocyte cKO mice after LPS administration showing reduced LCAT protein in astrocyte cKO mice ( n = 7). (R) Representative images of TNFRSF25 staining in the cortex of control and astrocyte cKO mice after LPS administration. Signal intensity quantification of TNFRSF25 staining ( n = 4 mice per genotype). * p < 0.05; scale bars indicate 200 μm. Data points are normalized to the mean of the control group. Error bars depict the SEM. Statistical comparisons were performed with the one-tailed Welch’s t test.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: RNA Sequencing, Control, Expressing, Marker, Western Blot, Staining, One-tailed Test

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Schematic diagram illustrating the elements required for the designed AAV for astrocyte-specific Chd8 editing in vivo via CRISPR-SaCas9. (B) Diagram for the simultaneous AAV injection and stab-wound injury with analysis of reactive gliosis performed at 7 days post-injection. (C) Representative images near the needle track from control mice (Scramble-AAV injected). CHD8 is detectable in astrocytes (SOX9 + ) expressing SaCas9 (HA tag + ). (D) Representative images near the needle track from mice injected with the Chd8 -cKO AAVs. CHD8 is undetectable in the majority of HA + and SOX9 + astrocytes (white arrows), while fewer HA + and SOX9 + astrocytes still show CHD8 expression (yellow arrows). (E and F) GFAP and Iba1 staining of astrocytes and microglia, respectively, after stab-wound injury and AAV injection in the Scramble-AAV (E) and Chd8 -cKO-AAV groups (F). (G) Decreased area occupied by GFAP + astrocytes in the Chd8 -cKO-AAV mice. (H) Quantification of the area occupied by Iba1 + microglia between control and Chd8 -cKO-AAV mice. In (C) and (D), scale bars indicate 20 μm; in (E) and (F), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified in (G) and (H). The dashed lines in merged images indicate the needle track of the injury. In (G) and (H), statistical comparisons were performed with two-way ANOVA; ** p < 0.01. Data points indicate n = 3 mice per group. Data are normalized to the means of the ipsilateral site in the control group.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: In Vivo, CRISPR, Injection, Control, Expressing, Staining

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: Figure 1. Reduced reactive gliosis in the global Chd8-cKO mice in the stab-wound injury model (A) Genetic strategy targeting exon 4 of the Chd8 gene. The exon was flanked with loxP sites to excise the loci, resulting in a frameshift mutation that disrupts the production of the CHD8 protein after Cre excision.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CHD8 Rabbit polyclonal 1 to 500 Abcam ab84527 CHD8 Rabbit polyclonal 1 to 1000 Novus Biologicals NB 100-60418 GFAP Guinea pig polyclonal antiserum 1 to 1000 Synaptic Systems 173 004 Iba1 Chicken polyclonal 1 to 1000 Synaptic Systems 234 006 Iba1 Rabbit Monoclonal 1 to 1000 Abcam ab178846 BrdU Mouse Monoclonal 1 to 200 Sigma-Aldrich MAB4072 Ki67 Rabbit Monoclonal 1 to 500 Invitrogen MA5-14520 tdTomato Rat Monoclonal 1 to 500 Kerafast EST203 LCAT Rabbit polyclonal 1 to 200 for IHC, 1 to 500 for WB Invitrogen PA5-22965 DR3/TNFRSF25 Mouse Monoclonal 1 to 200 for IHC,1 to 500 for WB R&D Systems MAB943 SOX9 Goat antiserum 1 to 200 R&D Systems AF3075 HA tag Rat IgG1 1 to 200 Roche 11867431001 goat-anti-mouse IRDye680 Li-COR Biosciences 926–68070 donkey-anti-rabbit IRDye 800CW Li-COR Biosciences 926–32213 Bacterial and virus strains pAAV2/5 Addgene Addgene # 104964 pAdDeltaF6 helper Addgene Addgene # 112867 pAAV-U6-scramble-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Rosa26-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Chd8-GfaABC1D-SaCas9-HA vector This paper N/A Chemicals, peptides, and recombinant proteins Tamoxifen Sigma T5648 OptiPrep Density Gradient Sigma D1556 DAPI Sigma D9542 Antifade mounting medium Vector Laboratories VECTH1000 BrdU Sigma B5002 Lipopolysaccharide Sigma L2880 Cas9 protein NEB M0646T Critical commercial assays RNA synthesis kit NEB E2040S LA-PCR Takara RR002A RNeasy mini kit QIAGEN 74104 iScript cDNA Synthesis Kit Bio-Rad 1725035 iQ SYBR Green Super mix kit Bio-Rad 1708882 TruSeq ribodepleted mRNA stranded for library preparation Illumina 20020594 ATAC-Seq Kit Active Motif 53150 Adult Brain Dissociation Kit, mouse and rat Miltenyi Biotec 130-107-677 Anti-CD11b Magnetic Microbeads kit for microglia Miltenyi Biotec 130–093-634 Anti-ACSA-2 MicroBead Kit for astrocyte Miltenyi Biotec 130-097-678 Deposited data Tissue Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271650 Microglia Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271651 Astrocyte ATAC-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271652 (Continued on next page) Cell Reports 43, 114637, August 27, 2024 21

Techniques: Mutagenesis

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: Figure 2. Reduced reactive gliosis in astrocyte Chd8-cKO mice in the stab-wound injury model (A) Strategy for tamoxifen-induced, astrocyte-specific Chd8 cKO utilizing the Aldh1l1-CreERT2 line. Mice were crossed with the Ai14-tdTomato reporter line to visualize recombined cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CHD8 Rabbit polyclonal 1 to 500 Abcam ab84527 CHD8 Rabbit polyclonal 1 to 1000 Novus Biologicals NB 100-60418 GFAP Guinea pig polyclonal antiserum 1 to 1000 Synaptic Systems 173 004 Iba1 Chicken polyclonal 1 to 1000 Synaptic Systems 234 006 Iba1 Rabbit Monoclonal 1 to 1000 Abcam ab178846 BrdU Mouse Monoclonal 1 to 200 Sigma-Aldrich MAB4072 Ki67 Rabbit Monoclonal 1 to 500 Invitrogen MA5-14520 tdTomato Rat Monoclonal 1 to 500 Kerafast EST203 LCAT Rabbit polyclonal 1 to 200 for IHC, 1 to 500 for WB Invitrogen PA5-22965 DR3/TNFRSF25 Mouse Monoclonal 1 to 200 for IHC,1 to 500 for WB R&D Systems MAB943 SOX9 Goat antiserum 1 to 200 R&D Systems AF3075 HA tag Rat IgG1 1 to 200 Roche 11867431001 goat-anti-mouse IRDye680 Li-COR Biosciences 926–68070 donkey-anti-rabbit IRDye 800CW Li-COR Biosciences 926–32213 Bacterial and virus strains pAAV2/5 Addgene Addgene # 104964 pAdDeltaF6 helper Addgene Addgene # 112867 pAAV-U6-scramble-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Rosa26-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Chd8-GfaABC1D-SaCas9-HA vector This paper N/A Chemicals, peptides, and recombinant proteins Tamoxifen Sigma T5648 OptiPrep Density Gradient Sigma D1556 DAPI Sigma D9542 Antifade mounting medium Vector Laboratories VECTH1000 BrdU Sigma B5002 Lipopolysaccharide Sigma L2880 Cas9 protein NEB M0646T Critical commercial assays RNA synthesis kit NEB E2040S LA-PCR Takara RR002A RNeasy mini kit QIAGEN 74104 iScript cDNA Synthesis Kit Bio-Rad 1725035 iQ SYBR Green Super mix kit Bio-Rad 1708882 TruSeq ribodepleted mRNA stranded for library preparation Illumina 20020594 ATAC-Seq Kit Active Motif 53150 Adult Brain Dissociation Kit, mouse and rat Miltenyi Biotec 130-107-677 Anti-CD11b Magnetic Microbeads kit for microglia Miltenyi Biotec 130–093-634 Anti-ACSA-2 MicroBead Kit for astrocyte Miltenyi Biotec 130-097-678 Deposited data Tissue Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271650 Microglia Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271651 Astrocyte ATAC-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271652 (Continued on next page) Cell Reports 43, 114637, August 27, 2024 21

Techniques:

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: Figure 3. Reduced proliferation and impaired morphological changes of reactive astrocytes in astrocyte Chd8-cKO mice (A and B) Representative images show reduced staining for proliferation markers (BrdU and Ki67) after stab-wound injury in astrocyte cKO mice (B) as compared to control mice (A). The dashed rectangle indicates the ROI used to quantify BrdU+ and Ki67+ cells in (C)–(F). (C) Decreased BrdU+ nuclei in the astrocyte cKO mice as shown by high-magnification images and quantification. (D) Reduced Ki67+ nuclei in the astrocyte cKO mice. (E) Decreased proliferation of tdTomato+ astrocytes from astrocyte cKO mice as shown by reduced tdTomato+/BrdU+ colocalized cells. (F) Decreased proliferation of tdTomato+ astrocytes from astrocyte cKO mice as shown by reduced tdTomato+/BrdU+ colocalized cells. (G and H) Representative skeletonized images of two astrocytes from control (G) and astrocyte cKO mice (H).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CHD8 Rabbit polyclonal 1 to 500 Abcam ab84527 CHD8 Rabbit polyclonal 1 to 1000 Novus Biologicals NB 100-60418 GFAP Guinea pig polyclonal antiserum 1 to 1000 Synaptic Systems 173 004 Iba1 Chicken polyclonal 1 to 1000 Synaptic Systems 234 006 Iba1 Rabbit Monoclonal 1 to 1000 Abcam ab178846 BrdU Mouse Monoclonal 1 to 200 Sigma-Aldrich MAB4072 Ki67 Rabbit Monoclonal 1 to 500 Invitrogen MA5-14520 tdTomato Rat Monoclonal 1 to 500 Kerafast EST203 LCAT Rabbit polyclonal 1 to 200 for IHC, 1 to 500 for WB Invitrogen PA5-22965 DR3/TNFRSF25 Mouse Monoclonal 1 to 200 for IHC,1 to 500 for WB R&D Systems MAB943 SOX9 Goat antiserum 1 to 200 R&D Systems AF3075 HA tag Rat IgG1 1 to 200 Roche 11867431001 goat-anti-mouse IRDye680 Li-COR Biosciences 926–68070 donkey-anti-rabbit IRDye 800CW Li-COR Biosciences 926–32213 Bacterial and virus strains pAAV2/5 Addgene Addgene # 104964 pAdDeltaF6 helper Addgene Addgene # 112867 pAAV-U6-scramble-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Rosa26-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Chd8-GfaABC1D-SaCas9-HA vector This paper N/A Chemicals, peptides, and recombinant proteins Tamoxifen Sigma T5648 OptiPrep Density Gradient Sigma D1556 DAPI Sigma D9542 Antifade mounting medium Vector Laboratories VECTH1000 BrdU Sigma B5002 Lipopolysaccharide Sigma L2880 Cas9 protein NEB M0646T Critical commercial assays RNA synthesis kit NEB E2040S LA-PCR Takara RR002A RNeasy mini kit QIAGEN 74104 iScript cDNA Synthesis Kit Bio-Rad 1725035 iQ SYBR Green Super mix kit Bio-Rad 1708882 TruSeq ribodepleted mRNA stranded for library preparation Illumina 20020594 ATAC-Seq Kit Active Motif 53150 Adult Brain Dissociation Kit, mouse and rat Miltenyi Biotec 130-107-677 Anti-CD11b Magnetic Microbeads kit for microglia Miltenyi Biotec 130–093-634 Anti-ACSA-2 MicroBead Kit for astrocyte Miltenyi Biotec 130-097-678 Deposited data Tissue Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271650 Microglia Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271651 Astrocyte ATAC-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271652 (Continued on next page) Cell Reports 43, 114637, August 27, 2024 21

Techniques: Staining, Control

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: Figure 6. CHD8 mediates chromatin accessibility changes during LPS-induced neuroinflammation (A) Flowchart of the experimental pipeline used to obtain astrocyte-enriched mouse brain samples for the subsequent ATAC-seq processing. (B) Genomic annotation enrichment for altered (increased and decreased) chromatin accessibility between saline-treated control mice and LPS-treated control mice. (C) Genomic annotation enrichment for altered (increased and decreased) chromatin accessibility between LPS-treated astrocyte cKO mice and LPS-treated control mice. (D) Heatmap display of ATAC-seq deviations in chromatin accessibility across the three conditions. (E) Heatmap representation of changes in ATAC-seq peaks near DEGs identified in our bulk RNA-seq experiments in Figure 5A. (F and G) Representative genome tracks showing loss of accessibility proximal to transcription start sites of the Basp1 gene identified in a previous study (F) and of the Lcat gene identified in our RNA-seq (G), in the astrocyte cKO samples.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CHD8 Rabbit polyclonal 1 to 500 Abcam ab84527 CHD8 Rabbit polyclonal 1 to 1000 Novus Biologicals NB 100-60418 GFAP Guinea pig polyclonal antiserum 1 to 1000 Synaptic Systems 173 004 Iba1 Chicken polyclonal 1 to 1000 Synaptic Systems 234 006 Iba1 Rabbit Monoclonal 1 to 1000 Abcam ab178846 BrdU Mouse Monoclonal 1 to 200 Sigma-Aldrich MAB4072 Ki67 Rabbit Monoclonal 1 to 500 Invitrogen MA5-14520 tdTomato Rat Monoclonal 1 to 500 Kerafast EST203 LCAT Rabbit polyclonal 1 to 200 for IHC, 1 to 500 for WB Invitrogen PA5-22965 DR3/TNFRSF25 Mouse Monoclonal 1 to 200 for IHC,1 to 500 for WB R&D Systems MAB943 SOX9 Goat antiserum 1 to 200 R&D Systems AF3075 HA tag Rat IgG1 1 to 200 Roche 11867431001 goat-anti-mouse IRDye680 Li-COR Biosciences 926–68070 donkey-anti-rabbit IRDye 800CW Li-COR Biosciences 926–32213 Bacterial and virus strains pAAV2/5 Addgene Addgene # 104964 pAdDeltaF6 helper Addgene Addgene # 112867 pAAV-U6-scramble-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Rosa26-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Chd8-GfaABC1D-SaCas9-HA vector This paper N/A Chemicals, peptides, and recombinant proteins Tamoxifen Sigma T5648 OptiPrep Density Gradient Sigma D1556 DAPI Sigma D9542 Antifade mounting medium Vector Laboratories VECTH1000 BrdU Sigma B5002 Lipopolysaccharide Sigma L2880 Cas9 protein NEB M0646T Critical commercial assays RNA synthesis kit NEB E2040S LA-PCR Takara RR002A RNeasy mini kit QIAGEN 74104 iScript cDNA Synthesis Kit Bio-Rad 1725035 iQ SYBR Green Super mix kit Bio-Rad 1708882 TruSeq ribodepleted mRNA stranded for library preparation Illumina 20020594 ATAC-Seq Kit Active Motif 53150 Adult Brain Dissociation Kit, mouse and rat Miltenyi Biotec 130-107-677 Anti-CD11b Magnetic Microbeads kit for microglia Miltenyi Biotec 130–093-634 Anti-ACSA-2 MicroBead Kit for astrocyte Miltenyi Biotec 130-097-678 Deposited data Tissue Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271650 Microglia Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271651 Astrocyte ATAC-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271652 (Continued on next page) Cell Reports 43, 114637, August 27, 2024 21

Techniques: Saline, Control, RNA Sequencing

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: Figure 7. CRISPR-SaCas9-mediated Chd8 editing through AAV in astrocytes mitigates reactive gliosis in the stab-wound injury model (A) Schematic diagram illustrating the elements required for the designed AAV for astrocyte-specific Chd8 editing in vivo via CRISPR-SaCas9. (B) Diagram for the simultaneous AAV injection and stab-wound injury with analysis of reactive gliosis performed at 7 days post-injection. (C) Representative images near the needle track from control mice (Scramble-AAV injected). CHD8 is detectable in astrocytes (SOX9+) expressing SaCas9 (HA tag+). (D) Representative images near the needle track from mice injected with the Chd8-cKO AAVs. CHD8 is undetectable in the majority of HA+ and SOX9+ astrocytes (white arrows), while fewer HA+ and SOX9+ astrocytes still show CHD8 expression (yellow arrows). (E and F) GFAP and Iba1 staining of astrocytes and microglia, respectively, after stab-wound injury and AAV injection in the Scramble-AAV (E) and Chd8-cKO-AAV groups (F). (G) Decreased area occupied by GFAP+ astrocytes in the Chd8-cKO-AAV mice. (H) Quantification of the area occupied by Iba1+ microglia between control and Chd8-cKO-AAV mice. In (C) and (D), scale bars indicate 20 mm; in (E) and (F), scale bars indicate 500 mm. The dashed rectangle indicates the ROIs that were quantified in (G) and (H). The dashed lines in merged images indicate the needle track of the injury. In (G) and (H), statistical comparisons were performed with two-way ANOVA; **p < 0.01. Data points indicate n = 3 mice per group. Data are normalized to the means of the ipsilateral site in the control group.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CHD8 Rabbit polyclonal 1 to 500 Abcam ab84527 CHD8 Rabbit polyclonal 1 to 1000 Novus Biologicals NB 100-60418 GFAP Guinea pig polyclonal antiserum 1 to 1000 Synaptic Systems 173 004 Iba1 Chicken polyclonal 1 to 1000 Synaptic Systems 234 006 Iba1 Rabbit Monoclonal 1 to 1000 Abcam ab178846 BrdU Mouse Monoclonal 1 to 200 Sigma-Aldrich MAB4072 Ki67 Rabbit Monoclonal 1 to 500 Invitrogen MA5-14520 tdTomato Rat Monoclonal 1 to 500 Kerafast EST203 LCAT Rabbit polyclonal 1 to 200 for IHC, 1 to 500 for WB Invitrogen PA5-22965 DR3/TNFRSF25 Mouse Monoclonal 1 to 200 for IHC,1 to 500 for WB R&D Systems MAB943 SOX9 Goat antiserum 1 to 200 R&D Systems AF3075 HA tag Rat IgG1 1 to 200 Roche 11867431001 goat-anti-mouse IRDye680 Li-COR Biosciences 926–68070 donkey-anti-rabbit IRDye 800CW Li-COR Biosciences 926–32213 Bacterial and virus strains pAAV2/5 Addgene Addgene # 104964 pAdDeltaF6 helper Addgene Addgene # 112867 pAAV-U6-scramble-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Rosa26-GfaABC1D-SaCas9-HA vector This paper N/A pAAV-U6-Chd8-GfaABC1D-SaCas9-HA vector This paper N/A Chemicals, peptides, and recombinant proteins Tamoxifen Sigma T5648 OptiPrep Density Gradient Sigma D1556 DAPI Sigma D9542 Antifade mounting medium Vector Laboratories VECTH1000 BrdU Sigma B5002 Lipopolysaccharide Sigma L2880 Cas9 protein NEB M0646T Critical commercial assays RNA synthesis kit NEB E2040S LA-PCR Takara RR002A RNeasy mini kit QIAGEN 74104 iScript cDNA Synthesis Kit Bio-Rad 1725035 iQ SYBR Green Super mix kit Bio-Rad 1708882 TruSeq ribodepleted mRNA stranded for library preparation Illumina 20020594 ATAC-Seq Kit Active Motif 53150 Adult Brain Dissociation Kit, mouse and rat Miltenyi Biotec 130-107-677 Anti-CD11b Magnetic Microbeads kit for microglia Miltenyi Biotec 130–093-634 Anti-ACSA-2 MicroBead Kit for astrocyte Miltenyi Biotec 130-097-678 Deposited data Tissue Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271650 Microglia Bulk RNA-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271651 Astrocyte ATAC-seq data NCBI - Gene Expression Omnibus GEO accession number: GSE271652 (Continued on next page) Cell Reports 43, 114637, August 27, 2024 21

Techniques: CRISPR, In Vivo, Injection, Control, Expressing, Staining