Journal: Genome Biology

Article Title: Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9

doi: 10.1186/s13059-020-01973-w

Figure Lengend Snippet: Multiplexed CAPTURE of locus-specific long-range DNA interactions. a Schematic of multiplexed analysis of locus-specific chromatin interactions by the redesigned CAPTURE2.0 system containing the C-terminal biotin-tagged dCas9 (dCas9-CBio) and sgRNA. Major steps of the CAPTURE-3C-seq method are shown. b Schematic of dCas9-mediated multiplexed capture of the human β-globin LCR. c Genome-wide analysis of dCas9 binding in cells expressing LCR-targeting sgRNAs (sgLCR) or non-targeting sgGal4. Data points for the sgRNA target regions are shown by arrowheads, and the predicted off-targets are shown as red dots. The x - and y -axes denote the log2 mean read counts and the log2 ratio of read counts in sgLCR and sgGal4 samples from N = 2 and 4 CAPTURE-ChIP-seq experiments, respectively. d Genome-wide differential gene expression analysis was performed using RNA-seq in K562 cells expressing dCas9-CBio with sgLCR or wild-type (WT) K562 cells. The β-like globin genes are indicated by colored data points. Pearson correlation coefficient ( R ) value is shown ( N = 2 RNA-seq experiments). e Browser view of LCR-mediated long-range interactions (chr11: 5,222,424-5,323,623; hg19) is shown. Contact profiles including the density map and interactions (or loops) for the dCas9-captured LCR or the resolved individual HS regions are shown. The statistical significance of interactions was determined by the Bayes factor (BF) and indicated by the color scale bars. DHS, ChIP-seq, RNA-seq, ChromHMM, CAPTURE-ChIP-seq (sgLCR), and ChIA-PET (CTCF and RNAPII) data are shown for comparison

Article Snippet: Human K562 cells were obtained from ATCC and cultured in an IMDM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: 3C-Seq, Genome Wide, Binding Assay, Expressing, ChIP-sequencing, Gene Expression, RNA Sequencing, ChIA Pet Assay, Comparison

Journal: Genome Biology

Article Title: Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9

doi: 10.1186/s13059-020-01973-w

Figure Lengend Snippet: Multiplexed CAPTURE of erythroid super-enhancers. a Schematic of the multiplexed analysis of erythroid SEs. SEs were identified by ROSE using H3K27ac ChIP-seq signal in K562 cells. Schematic of sgRNA design is shown. Pie charts on the left show the distribution of the captured SEs and constituent enhancers at intragenic, intergenic, or both regions. Pie charts on the right show the numbers and percentages of captured SEs and constituent enhancers (green color). b Genome-wide analysis of dCas9 binding in cells expressing SE-targeting sgRNAs (sgSE) or non-targeting sgGal4. Data points for the sgRNA captured SEs are shown as green. The x - and y -axes denote the log2 mean read counts and the log2 ratio of read counts in sgSE and sgGal4 samples from N = 2 and 4 CAPTURE-ChIP-seq experiments, respectively. c Genome-wide differential gene expression analysis was performed using RNA-seq in K562 cells expressing dCas9-CBio with sgSE or WT K562 cells. Data points for SE target genes and other genes are shown as red and gray, respectively. Pearson correlation coefficient ( R ) value is shown ( N = 2 RNA-seq experiments). d Analysis of SE-mediated long-range interactions by categorizing all interactions into SEs to gene promoters (SE-P), SEs to gene bodies (SE-G), and SEs to other genomic regions (SE-O). Schematic of SE-mediated interactions is shown on the top. The interaction frequency was calculated by the normalized PETs per kilobase of captured DNA sequences. Boxes show the median of the data and quartiles, and whiskers extend to 1.5× of the interquartile range. P values were calculated by a two-sided Kolmogorov-Smirnov (K-S) test. e Pie chart shows the fractions of the captured SE-mediated interactions between SEs and gene targets. f A representative locus is shown for the single SE to single gene interactions (SE137). Contact profiles including the density map and interactions for the dCas9-captured SE region (red bar) are shown. The statistical significance of interactions was determined by the Bayes factor (BF) and indicated by the color scale bars. DHS, ChIP-seq, ChromHMM, ChIA-PET (CTCF and RNAPII), and in situ Hi-C data are shown for comparison. g A representative locus is shown for the single SE to multiple genes (SE43). h A representative locus is shown for the multiple SEs to multiple genes (SE41 and SE42)

Article Snippet: Human K562 cells were obtained from ATCC and cultured in an IMDM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: ChIP-sequencing, Genome Wide, Binding Assay, Expressing, Gene Expression, RNA Sequencing, ChIA Pet Assay, In Situ, Hi-C, Comparison

Journal: Genome Biology

Article Title: Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9

doi: 10.1186/s13059-020-01973-w

Figure Lengend Snippet: Hierarchical organization of super-enhancers identified by multiplexed CAPTURE. a Schematic of the hierarchical structure of SEs based on constituent enhancer-mediated long-range chromatin interactions. b Identification of hierarchical SEs by the H-score computational metric. c A representative locus is shown for a hierarchical SE containing the hub enhancer. Contact profiles including the density map, interactions between enhancers, and all interactions for the dCas9-captured SE region (red bar) are shown. The identified hub and non-hub enhancers are depicted by green (hub) and red (non-hub) lines, respectively. d A representative locus is shown for a non-hierarchical SE without hub enhancer. e Chromatin landscapes at hub, non-hub enhancers, and non-hierarchical SEs in K562 cells. Spatial distribution of DHS, histone marks (H3K4me1 and H3K27ac), TFs (p300, GATA1, and TAL1), RNAPII, and chromatin structure factors (CTCF, SMC3, and RAD21) is shown in hub ( N = 42) and non-hub enhancers ( N = 260) and non-hierarchical SEs ( N = 117). P values were calculated using the two-sample Kolmogorov-Smirnov (K-S) test. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s. not significant

Article Snippet: Human K562 cells were obtained from ATCC and cultured in an IMDM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage

doi: 10.1083/jcb.201612131

Figure Lengend Snippet: Recruitment of Dicer to DNA double-strand breaks in Asi SI-ER U2OS cells. (A) Structure of genomic loci assessed by quantitative RT-PCT (left) and genome-wide Asi SI-ER target site distribution; n, number of predicted Asi SI-ER target sites (right); fwd/rev, forward/reverse. (B) Immunoblots detecting total Dicer (A-2) and γH2A.X after induction of DNA double-strand breaks (DSBs). 4OHT, 4-hydroxytamoxifen. Immunoblots were quantified using ImageJ. (C) Confocal imaging of phosphorylated Dicer (p-DCR-1) and γH2A.X (top). All quantifications represent number of cells exhibiting shown phenotype. Quantification using ImageJ RGB profiler (bottom). (D) ChIP analysis showing Dicer occupancy at DSBs DS1/2 in wild-type and Asi SI-ER U2OS cells using site-specific primers. GAPDH, control locus. *, P < 0.05; error bars, means ± SEM of three biological replicates. (E) ChIP analysis showing Dicer occupancy at DS1 in absence or presence of recombinant RNase III preincubation. *, P < 0.05; error bars, means ± SEM of three biological replicates. (F) ChIP-seq signal upon +4OHT incubation at 200 γH2A.X-positive/negative genic sites after removal of duplicate reads. A rolling mean of 1 kb was applied after removal of 2% of the top and bottom values. Shadow, rolling SD. (G) Snapshot showing Dicer binding at genic Asi SI target site upstream of TRIM37 before (4OHT − ) and after (4OHT + ) DNA damage. Red box, proximal region to Asi SI site.

Article Snippet: RNA digestions were performed using RNase III (1 U; New England Biolabs, Inc.) for 1 h at 37°C.

Techniques: Genome Wide, Western Blot, Imaging, Recombinant, ChIP-sequencing, Incubation, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage

doi: 10.1083/jcb.201612131

Figure Lengend Snippet: Nuclear accumulation of S1016 phosphorylated Dicer upon DNA damage in HEK293 cells. (A) Schematic of human Dicer isoform 1 ( NP_001258211.1 ) domain structure and positions of assessed phosphorylated residues. DUF283, domain of unknown function; PAZ, Piwi/Argonaute/Zwille; RIIIa/b, RNase III a/b; dsRBD, double-stranded RNA binding domain. (B) Confocal imaging of RFP-tagged Dicer constructs expressed in wild-type HEK293 cells. All quantifications represent the number of cells that have the shown phenotype. (C) Relative quantification of (B). Bars, mean ratio (nuclear/cytoplasmic RFP) normalized to the background, n > 45. (D) Absolute quantification of (B). Bars, mean number of cells with nuclear RFP signal, n > 45. *, P < 0.05.

Article Snippet: RNA digestions were performed using RNase III (1 U; New England Biolabs, Inc.) for 1 h at 37°C.

Techniques: RNA Binding Assay, Imaging, Construct

Journal: eLife

Article Title: Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington’s disease

doi: 10.7554/eLife.89782

Figure Lengend Snippet: ( A ) HEK293 cells were treated with empty vector (EV) or candidate BE strategies such as BE4max-gRNA 1 (gRNA 1) and BE4max-gRNA 2 (gRNA 2) for RNAseq analysis. MiSeq analysis was also performed to judge the levels of CAG-to-CAA conversion. ****, p-value<0.0001 by Student’s t-test (n=4). ( B ) Confirming the lack of significantly altered genes in BE4max-gRNA 1 or BE4max-gRNA 2, we compared all BE-treated samples (n=8) with all EV-treated samples (n=4) to increase the power in the RNAseq differential gene expression analysis. Each circle in the volcano plot represents a gene analyzed in the RNAseq; HTT is indicated by a filled red circle. A red horizontal line represents false discovery rate of 0.05, showing that none was significantly altered by candidate BE strategies. ( C ) We also compared two groups of randomly assigned samples (six samples vs. six samples) to understand the shape of the volcano plot when there were no significant genes.

Article Snippet: Subsequently, genomic DNA for MiSeq analysis and cell pellets for RNAseq analysis were generated from replica plates genome-wide RNAseq analysis (Tru-Seq strand-specific large insert RNAseq) was performed by the Broad Institute.

Techniques: Plasmid Preparation, Gene Expression

Journal: eLife

Article Title: Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington’s disease

doi: 10.7554/eLife.89782

Figure Lengend Snippet: HEK293 cells were treated with empty vector (EV), or candidate base editing (BE) strategies such as BE4max-gRNA 1 ( A ) and BE4max-gRNA 2 ( B ). Subsequently, DNA samples and RNA samples were collected for MiSeq analysis and RNAseq analysis to evaluate the levels of on-target conversion and changes in transcriptome (n=4), respectively. The most significant gene in cells treated with BE4max-gRNA 2 (panel B) was HSD3B1 , which was not significant by false discovery rate of 0.05 (red lines).

Article Snippet: Subsequently, genomic DNA for MiSeq analysis and cell pellets for RNAseq analysis were generated from replica plates genome-wide RNAseq analysis (Tru-Seq strand-specific large insert RNAseq) was performed by the Broad Institute.

Techniques: Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: The Polycomb protein RING1B enables estrogen-mediated gene expression by promoting enhancer–promoter interaction and R-loop formation

doi: 10.1093/nar/gkab723

Figure Lengend Snippet: RING1B promotes R-loop formation at ERα target genes via direct participation in their transcription. ( A ) RNA-seq volcano plot depicting one significantly downregulated and 11 significantly upregulated genes ( P -value < 0.05, FC > 1.5) in parental T47D cells before and after 4′ of E2 stimulation; n = 2. ( B ) RT-qPCR measuring levels of GREB1 , FMN1 and FKBP4 nascent transcripts in T47D and MCF7 shCTR and shRING1B cells before and after 45′ of E2 stimulation. shCTR – RT and shRING1B – RT are RNA samples subjected to the cDNA conversion process in the absence of reverse transcriptase. Enrichment detected in these samples represent genomic DNA contamination. Error bars represent the standard deviation of three biological replicates. * P -value < 0.05, ** P -value < 0.01, one-tailed paired t -test. ( C ) RNA-seq heatmap depicting expression of GREB1 , FMN1 , and FKBP4 in T47D shCTR, shRING1B, as well as shRING1B cells rescued with wildtype (WT), nucleosome-binding mutant (R98A) and a catalytic-dead mutant (I53A) RING1B; n = 2. ( D ) CBX4 and IgG, as a negative control, ChIP-qPCR of RING1B/ERα co-bound sites before and after 45 min of E2 administration; n = 2. ( E ) Endogenous RING1B immunoprecipitation with nuclear extracts after crosslinking with 1% FA for 10 min. Proteins bound to RING1B were identified by LC-MS/MS, and enrichment was calculated based on fold change over IgG enrichment and P -value < 0.05. IgG was used as a negative control. Experiments were performed in three biological replicates. Proteins labeled in green are PRC1 subunits. Proteins labeled in red are new RING1B-associated proteins. ( F ) Average DRIP-seq signals in parental T47D cells before and after 45′ of E2 stimulation at ERα and RING1B co-bound sites (top) and genes (bottom). No signal was detected in samples treated with RNase H, indicating that the signal observed was specific for R-loops. ( G ) T47D shCTR and shRING1B DRIP-seq signal before and after 45 min of E2 at the FMN1 and the ADAMTSL5 genes. ( H ) Average genome-wide DRIP-seq signal (left) and with respect to genes that contain R-loops (right). ( I) Average DRIP-seq signal at all RING1B and ERα co-bound sites (left) and genes (right) that contain R-loops.

Article Snippet: DNA concentration was measured using a nanodrop and 10 μg was digested with 4 μl of RNase H (NEB M0297L), 20 μl of RNase H buffer and water up to 200 μl for 4 h at 37°C to produce the RNase H treated negative control.

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Standard Deviation, One-tailed Test, Expressing, Binding Assay, Mutagenesis, Negative Control, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Labeling, Genome Wide

Journal: Cell

Article Title: Regulation of the RNAPII Pool Is Integral to the DNA Damage Response

doi: 10.1016/j.cell.2020.02.009

Figure Lengend Snippet:

Article Snippet: RNase-Free DNase Set , QIAGEN , 79254.

Techniques: Virus, Recombinant, Protease Inhibitor, Membrane, SYBR Green Assay, Reverse Transcription, Ubiquitin Proteomics, Genome Wide, Knock-In, Mutagenesis, Selection, Software

Journal: Nature Communications

Article Title: Reconstructing clonal tree for phylo-phenotypic characterization of cancer using single-cell transcriptomics

doi: 10.1038/s41467-023-36202-y

Figure Lengend Snippet: Coverage statistics for 10X vs Smart-Seq3 on HGSOC data

Article Snippet: Next, we investigate the applicability of PhylEx on widely available 10X Genomics scRNA-seq data (referred to as 10X for brevity).

Techniques:

Journal: Molecular therapy. Methods & clinical development

Article Title: PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease.

doi: 10.1016/j.omtm.2022.08.005

Figure Lengend Snippet: Figure 3. High levels of mutant HTT specificity supported by RNA-seq analysis Allele-specific expression (ASE) analysis was performed to evaluate the levels of allele specificity of our TP-CRISPR strategies. HD subjects with the most frequent diplotype (i.e., hap.01 and hap.08) are heterozygous at 10 exonic SNPs. Thus, we performed ASE using those 10 exonic SNP sites. Alleles of those 10 exonic SNPs on the mutant and normal HTT were based on our haplotype definitions and previous sequencing analysis. We counted the alleles on the mutant and normal HTT for a given SNP site, and then calculated average values. (A) Mean allele counts of 10 heterozygous exonic SNPs on the mutant HTT are summarized. Boxes on the left and right represent the distribution of alleles on the mutant HTT in EV-treated and targeted clonal lines, respectively. Student’s t test was performed (nominal p value, 3.53e6). (B) The same analysis approach was applied to alleles of 10 heterozygous exonic SNPs that are on the normal HTT. Boxes on the left and right represent the distribution of alleles in EV-treated and targeted clonal lines, respectively. Student’s t test was performed (nominal p value, 0.67). Each box shows maximum, 75%, 50% (median), 75% quartile, and minimum.

Article Snippet: Then, genome-wide RNA-seq analysis was performed by the Broad Institute.

Techniques: Mutagenesis, RNA Sequencing, Expressing, CRISPR, Sequencing

Journal: Molecular therapy. Methods & clinical development

Article Title: PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease.

doi: 10.1016/j.omtm.2022.08.005

Figure Lengend Snippet: Figure 4. On-target gene specificity supported by RNA-seq analysis To increase the sensitivity and the power to detect any small but significantly altered genes, we combined 10 targeted clonal lines targeted by L4-R4 and 10 clonal lines targeted by L4-R6 gRNA combinations to be compared with 12 EV-treated controls. (A) Significance values (un- corrected p values on the y axis) were compared with log2 (fold-change) (x axis) to highlight significantly altered genes. A horizontal and a vertical line represent Bonfer- roni-corrected significance and zero fold-change, respectively. (B) Expression levels of total HTT (mutant plus normal) based on the DGE analysis are summarized in a boxplot. Total HTT levels were decreased by 38% in TP-CRISPR targeted clonal lines.

Article Snippet: Then, genome-wide RNA-seq analysis was performed by the Broad Institute.

Techniques: RNA Sequencing, Expressing, Mutagenesis, CRISPR

Journal: Cell

Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

doi: 10.1016/j.cell.2013.03.012

Figure Lengend Snippet: Genome-wide Tfe3-Target Determination Identifies Esrrb as a Downstream Effector of Flcn-Fnip1/2-Tfe3 (A) Gene tracks of loci identified by Tfe3 ChIP-Seq with control (ctrl.2), Flcn shRNA (Flcn.4), and Tfe3 shRNA (Tfe3.3) cell lines, overlaid with Nanog-, Oct4- and Tcf3-bound regions. (B) ESCs expressing indicated constructs were treated for 3 hr with 0.1 μM Tam in 2i. Average mRNA fold changes relative to EtOH treatment and SD are from two independent experiments with three different cell lines per genotype. ( ∗ ) indicates Student’s t test values < 0.005. (C) Average mRNA changes in ESCs transfected with the indicated siRNA combinations. Average relative expression normalized to no and negative siRNA treatments and SD are of three independent experiments. ( ∗ ) indicates Student’s t test values < 0.05. (D) O4GIP ESCs expressing Tfe3-ERT2 were transfected with indicated siRNAs, differentiated for 3 days in the presence of 0.1 μM Tam or EtOH, and switched back to 2i with puromycin selection, and remaining ESC colonies were quantified with a cell-viability assay. Average fold changes over negative siRNA-transfected, EtOH-treated cells and SD are from two independent experiments. ( ∗∗ ) and ( ∗ ) indicate Student’s t test values < 0.001 and 0.03, respectively. (E) Commitment of O4GIP cells transfected with indicated siRNA combinations, including two independent Esrrb siRNAs. Exit from pluripotency was quantified with a cell-viability assay and normalized to negative siRNA treatment. Average and SD are of two technical replicates. See also Figure S6 .

Article Snippet: ChiP-Seq libraries were generated using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB) using 10 amplification cycles.

Techniques: Genome Wide, ChIP-sequencing, shRNA, Expressing, Construct, Transfection, Selection, Viability Assay