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: 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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: Dosage-sensitive effect of CHD8 deletion on transcription in ESCs. (A) The gnomAD human gene constraint scores (missense and probability of intolerance to heterozygous pLoF variation [pLI] for all genes with CHD8 labeled). (B) Schematic showing the CHD8 CRISPR-mediated deletion approach and Western blot of CHD8 levels in Chd8+/− and Chd8−/− ESC lines. (C) Doubling time of WT, Chd8+/−, and Chd8−/− cells. Asterisks denote P < 0.05 (*). Values are expressed as the mean ± SE, n = 3. (D) Alkaline phosphatase staining of WT, Chd8+/−, and Chd8−/− ESCs in 2i media. Magnification: 200×. (E) Venn diagrams depicting overlap of DEGs in Chd8+/− and Chd8−/− ESCs. Genes with increased expression are on top, and genes with decreased expression are on the bottom (FDR < 0.05). (F) Heatmap showing dosage-sensitive transcriptional response for genes that are differentially expressed in Chd8−/−. (G) GO terms for DEGs (padj < 0.05).

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Labeling, CRISPR, Western Blot, Staining, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 deletion results in up-regulation of neuronal genes and Sox TFs during differentiation into NPCs. (A) Schematic showing the differentiation of WT, Chd8+/−, and Chd8−/− ESCs into NPCs. (B) Venn diagrams depicting overlap of DEGs in Chd8+/− and Chd8−/− NPCs. Genes with increased gene expression are on top, and genes with decreased expression are on bottom (FDR < 0.05). (C) Heatmap showing dosage sensitive transcriptional response for genes that are differentially expressed in Chd8−/−. (D) GO terms for DEGs. (E and F) Volcano plots of RNA-seq data for CHD8+/− and CHD8−/−. DEGs are highlighted in red (FDR < 0.05), and genes involved with mature neuronal development and Sox TFs are labeled. Genes labeled with yellow background were stained by IF in G. (G) Representative IF staining of day 8 NPCs, with antibodies listed on left side and respective genotypes on top (n = 2 biological replicates with similar results). Magnification: 200×.

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Gene Expression, Expressing, RNA Sequencing, Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 regulates accessibility at key regions in ESCs and during differentiation to NPCs. (A) Percentage of increased (blue) and decreased (orange) peaks for Chd8−/− ESCs. (B) Genomic annotation enrichment for increased and decreased sites for Chd8−/− ESC ATAC-seq datasets. (C) Percentage of increased (blue) and decreased (orange) peaks for Chd8−/− NPCs. (D) Genomic annotation enrichment for increased and decreased sites for Chd8+/−and Chd8−/− NPC ATAC-seq datasets. (E) Genome tracks showing gain of accessibility at Grb10 locus in ESCs. (F) Genome tracks showing loss of accessibility at Basp1 locus in NPCs.

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 cooperates with Sox TFs to regulate accessibility and transcription. (A) Heatmap representation of ATAC-seq chromVAR bias-corrected deviations in the 50 most variable TF motifs across ESC WT and Chd8−/− ATAC-seq replicates. (B) Heatmap representation of ATAC-seq chromVAR bias-corrected deviations in the 50 most variable TF motifs across NPC WT and Chd8−/− ATAC-seq replicates. (C) Western blot showing co-IP of CHD8 with SOX2 in ESCs (antibodies used are the following: SOX2 A: sc-365823; SOX2 B: AB5603). (D) Heat map displaying CHD8 ChIP-seq at SOX2 peaks (41) (Left) and SOX2 ChIP-seq at CHD8 peaks (Right).

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Western Blot, Co-Immunoprecipitation Assay, ChIP-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: (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: Nucleic acids research

Article Title: CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

doi: 10.1093/nar/gkac1134

Figure Lengend Snippet: Figure 1. CHD8 suppression significantly impacts on histone H3K36me3 enrichment at transcriptional elongation sites. (A) Schematic representation of the study design and integrative approach used in this work. Human iPSC-derived NPCs (hiNPC) knocked down for CHD8 (Sh1-, Sh2- and Sh4- CHD8) and control hiNPCs (Sh-GFP and Sh-GFP2) (11), were analyzed via ChIP-seq for six histone marks representative of different chromatin regions: active promoters (H3K4me2 and H3K4me3), inactive promoters (H3K27me3), enhancers (H3K4me1 and H3K27ac) and actively transcribed regions (H3K36me3). ChIP-seq results were subsequently integrated with CHD8-binding sites and available transcriptomics (RNA-seq) datasets obtained from the same model system (11). (B) The heatmaps represent 10 different chromatin states (1, transcriptional initiation; 2, transcriptional elongation; 3, weakly

Article Snippet: For CHD8 immunoprecipitation, NB100-60417 and NB100-60418 (Novus Biotechnology), for hnRNPL D-5 D ow nloaded from https://academ ic.oup.com /nar/article/50/22/12809/6947080 by guest on 10 February 2024 (sc-48391, Santacruz) and for SETD2 (38633, SAB) were used, while rabbit IgG isotype control (10500C, Life) or mouse IgG (10400C, Invitrogen) were used as controls.

Techniques: Derivative Assay, Control, ChIP-sequencing, Binding Assay, RNA Sequencing

Journal: Nucleic acids research

Article Title: CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

doi: 10.1093/nar/gkac1134

Figure Lengend Snippet: Figure 3. CHD8 suppression-elicited reduction in H3K36me3 correlates with significant alterations in RNA AS. (A, B) Venn diagrams represent the overlap between genes losing H3K36me3 peaks following CHD8 knockdown (losing H3K36me3) and genes presenting altered AS events as detected by SUPPA

Article Snippet: For CHD8 immunoprecipitation, NB100-60417 and NB100-60418 (Novus Biotechnology), for hnRNPL D-5 D ow nloaded from https://academ ic.oup.com /nar/article/50/22/12809/6947080 by guest on 10 February 2024 (sc-48391, Santacruz) and for SETD2 (38633, SAB) were used, while rabbit IgG isotype control (10500C, Life) or mouse IgG (10400C, Invitrogen) were used as controls.

Techniques: Knockdown

Journal: Nucleic acids research

Article Title: CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

doi: 10.1093/nar/gkac1134

Figure Lengend Snippet: Figure 4. hnRNPL as a novel CHD8 interactor: bridging altered splicing to H3K36me3 enrichment. (A) Schematic representation of the MS/MS ex- perimental design and approach used in this work (figure created in BioRender.com). Nuclei from hiNPCs (11) were separated from the cytoplasmic fraction. The protein of interest was isolated from the nuclear lysate by specific primary antibodies followed by incubation with Sepharose beads. CHD8 immunoprecipitated proteins were then processed by in solution trypsin digestion prior to MS/MS analysis. (B) Representative western blot images depict immunoprecipitation by endogenous, full-length CHD8 in nuclear extracts by two different antibodies CHD8 NB100-60417 (CHD8 #17) and NB100- 60418 (CHD8 #18). A strong, reproducible enrichment compared with Input (Input, 15 g of nuclear lysate) and rabbit IgG control (IgG) is evident. High exp, 30 s; low exp = 4 s. (C) The volcano plots show CHD8-interacting proteins, significantly differentially enriched compared with IgG controls. Significantly enriched proteins are in blue, significantly depleted proteins in red and non-significant proteins in gray. The threshold for significance is set at a P-value of 0.05. Three independent experiments were averaged and analyzed together for each condition. CHD8, the more represented and enriched peptide with each of the two antibodies, was removed from the plots to optimize visualization of interactors. (D) Venn diagrams represent the overlap be- tween CHD8-interacting proteins identified by CHD8 #17 and CHD8 #18 antibodies. The analysis combines the statistically significant results from three independent biological replicates and two antibodies (Ab #17 A, B, C; Ab #18 A, B, C; see also Supplementary Figure S11). The number of proteins for each condition is indicated. The complete list of proteins is given in Supplementary Table S1. (E) Complete list of the 18 CHD8-interacting proteins identi- fied by CHD8 #17 and CHD8 #18 antibodies. (F) Representative western blot images from co-immunoprecipitation experiments demonstrate interaction between endogenous CHD8 and hnRNPL. Immunoprecipitations were conducted with the two antibodies (IP CHD8 #17 and IP CHD8 #18). A strong, reproducible CHD8 enrichment compared with Input (Input, 15 g of nuclear lysate, Input 5%, 0.75 g of nuclear lysate) and rabbit IgG control (IgG) is evident. Co-immunoprecipitation of hnRNPL is clearly visible at high exposure. CHD8 high exp, high exposure = 60 s; CHD8 low exp, low exposure = 20 s. HnRNPL high exp, high exposure = 240 s; CHD8 low exp, low exposure = 75 s. (G) Representative western blot images showing immunoprecipitation of endogenous CHD8 in the nuclear extract with different treatments: RNase A (RNaseA), EtBr or no treatment (NT). CHD8 high exp, high exposure = 30 s; CHD8 low exp, low exposure = 10 s. HnRNPL high exp, high exposure = 60 s; hnRNPL low exp, low exposure = 4 s. (H) Representative western blot images report co-immunoprecipitation between hnRNPL (anti-mouse) and SETD2 (anti-rabbit) antibodies. Endogenous hnRNPL interacts with SETD2, as demonstrated by enrichment over mouse IgG (IP IgG Mou) and INPUT (15 g of nuclear lysate). Reciprocal co-immunoprecipitation of endogenous SETD2 confirms the interaction, visible at high exposure, compared with IgG controls (IP IgG Rab). SETD2 high exp, high exposure = 20 s; SETD2 low exp, low exposure = 4 s. HNRNPL high exp, high exposure = 40 s; hNRNPL low exp, low exposure = 2 s.

Article Snippet: For CHD8 immunoprecipitation, NB100-60417 and NB100-60418 (Novus Biotechnology), for hnRNPL D-5 D ow nloaded from https://academ ic.oup.com /nar/article/50/22/12809/6947080 by guest on 10 February 2024 (sc-48391, Santacruz) and for SETD2 (38633, SAB) were used, while rabbit IgG isotype control (10500C, Life) or mouse IgG (10400C, Invitrogen) were used as controls.

Techniques: Tandem Mass Spectroscopy, Isolation, Incubation, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Human T-lymphocytes were reprogrammed into iPSCs. WT and CHD8 KD karyotypes were normal.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: CRISPR/Cas9-mediated CHD8 KD . (a) CRISPR/Cas9 target of 20 nucleotides (green box) and PAM site (green letters) adjacent to the CHD8 Ser62 codon was used for the single guide RNA. To facilitate homologous recombination, a donor DNA molecule was used consisting of left (HAL) and right (HAL) homology arms and loxP sites (blue arrows) flanking the EGFP cassette (EGFP cDNA with upstream splice acceptor sequence (SA), puromycin resistance gene (Puro), and CAG promoter). (b) EGFP facilitated manual selection of successfully targeted iPSC colonies under fluorescence microscopy. (c) This was confirmed by fluorescence-activated cell sorting.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques: CRISPR, Homologous Recombination, Sequencing, Selection, Fluorescence, Microscopy, FACS

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Gene expression analysis. (a) qRT-PCR showed that, compared to WT, CHD8 mRNA levels were reduced in KD cell lines by an average of 41% in iPSCs, 52% in NPCs, and 59% in neural cells. (b and c) Western blot analysis of NPCs showed that CHD8 protein was reduced by an average of 58%.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Effects of CHD8 KD on proliferation and differentiation. CHD8 KD caused (a) increased iPSC colony numbers, (b) increased NPC growth, and (c) suppression of spontaneous differentiation around the edges of iPSC colonies.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: NPC rosette formation. During the differentiation of iPSCs to NPCs, cells typically organize into rosettes by approximately two weeks. CHD8 KD delayed rosette formation by one week, or by approximately a third.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Neurite outgrowth. 12 days after NPCs were plated, CHD8 KD resulted in a significantly decreased neurite area (white arrows) per cell (nuclear) area.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Effect of CHD8 KD on the cell cycle. A significantly smaller percentage of CHD8 KD NPCs (58.84 ± 4.59) is found in the G0/G1 phase of the cell cycle compared to WT NPCs (72.16 ± 6.8, P = 0.007, t-test), and a larger percentage of CHD8 KD NPCs (21.90 ± 7.08) is found in the G2/M phases of the cell cycle compared to WT NPCs (12.10 ± 6.82, P = 0.057, t-test).

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Effects of CHD8 KD on cell composition. CHD KD resulted in: (a) decreased MAP2/GFAP ratio, (b) decreased fraction of VGLUT1+ cells, and (c) decreased percentage of SYN1+ cells.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques: