pen84 plasmid  (Addgene inc)

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Diagram of the CTCF-degron. b, Left: Targeting strategy for introducing in-frame mAID-GFP sequences into both Ctcf alleles. Cas9/sgRNAs and southern blotting probe are indicated. B: BglII. Right: Southern blotting confirmation of correctly targeted alleles ( n =3 biological repeats with similar results). c, d, Southern blotting confirmation of correctly targeted Rosa26 alleles in CTCF-degron ( c , n =3 biological repeats with similar results) and OsTir1-expressing ( d , left, n =2 biological repeats with similar results) lines following OsTir1-V5 targeting strategy shown in c . Western blotting on nuclear extracts of G1-arresed OsTir1-expressing cells with correctly targeted Rosa26 alleles confirms the constitutive expression of OsTir1-V5 protein ( d , right, n =2 biological repeats with similar results). Histone H3 was used as a loading control. e, Representative GFP signal-based flow-cytometry plots for CTCF-degron cells that are non-treated (NT) or treated with IAA (+IAA) for 6h (top) followed by 4-day STI-571 treatment (without or with IAA) (bottom). The parental cells were used as GFP negative controls. Three biological repeats with similar results. f, Fucci cell cycle assay of CTCF-degron cells without (NT) or with IAA treatment followed by 4-day STI-571 treatment. Representative flow-cytometry plots and average percentage ± s.d. of cells arrested in G1-stage at indicated condition ( n =3 biologically independent samples) are shown. The parental cells without Fucci were used as negative controls. g, Western blotting on nuclear extracts of G1-arrested cells as indicated using indicated antibodies ( n =3 biological repeats with similar results). h, Time-course cell viability assay for parental and IAA-treated CTCF-degron cells, following STI-571 treatment for G1-arrest (+STI). Average percentage ± s.d. of viable cells for each timepoint and under each condition was shown ( n =3 biologically independent samples). p values were calculated using unpaired two-tailed t -test. See Methods.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Southern Blot, Expressing, Western Blot, Control, Flow Cytometry, Cell Cycle Assay, Viability Assay, Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Diagram of joining between RAG-mediated breaks at initiating bona fide J H 4-RSS (blue triangle) and cryptic RSSs, mostly represented by CAC motifs (green triangles), across Igh locus, upon down-modulation of CTCF/CBEs (indicated by light purple arrowheads). Deletional and inversional joining is denoted by red (+) and blue (−) curves, respectively. b, Pooled HTGTS junction profiles across highlighted V H domains for deletional and inversional joining in RAG2-complemented, IAA-treated and G1-arrested parental ( n =4 biological repeats with similar results) and CTCF-degron ( n =6 biological repeats with similar results) cells. For presentation, all libraries from parental or CTCF-degron cells were pooled and normalized to 3,819 total off-target junctions. Note that the high-level deletional cryptic RSS joining signals (red bars) within IGCR1-J H region are off-scale on the y -axis for viewing of relatively lower level deletional cryptic RSS joining signals across the V H locus. The low-level, but reproducible, inversional joining events (blue bars) within IGCR1-J H region involve cryptic RSSs predominantly at IGCR1 and D H 3–2 locales, as described . The vast majority of deletional joining events across the V H locus are reproducible among replicates and involve, at a minimum the CAC of an RSS. Almost all of the handful of the very low-level inversional joining events interspersed across V H locus are not reproducible among replicates consistent with being background events. c, Average frequencies ± s.d. of deletional (left) and inversional (right) joining events within indicated regions in RAG2-complemented, IAA-treated and G1-arrested parental ( n =4 biological repeats with similar results) and CTCF-degron ( n =6 biological repeats with similar results) cells. Each of the biological library replicates was normalized to 415 off-target junctions for statistical analysis. p values were calculated using unpaired two-tailed t -test. NA: not applicable. See Methods.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a , Average 3C-HTGTS signal counts ± s.e.m. across the four V H domains in G1-arrested parental (top, n =3) and IAA-treated CTCF-degron (middle, n =3) v-Abl cells and RAG2-deficient mouse BM pro-B cells (bottom, n =2). In all panels, “n” indicates biological repeats. Spearman’s correlation coefficient (r) and p values determined by two-sided Spearman’s correlation test between data in middle and bottom panels are: r=0.86, p <2.2×10 -16 . Related low-level RC interactions across the parental cell V H locus and those of BM pro-B cells (r=0.63) and IAA-treated CTCF-degron cells (r=0.74) suggest low-level Igh loop extrusion in parental cells. b - d , Average V H utilization frequencies ± s.d. in RAG2-complemented, IAA-treated and G1-arrested parental ( b , n =4), CTCF-degron ( c , n =6) v-Abl cells and BM pro-B cells ( d , n =6). Average percentage ± s.d. of V H DJ H and DJ H rearrangements are indicated. Spearman’s correlation coefficient (r) and p values by two-sided Spearman’s correlation test between data in panels c and d are r=0.76, p<2.2×10 -16 . See and . e , Average utilization frequencies ± s.d. of indicated V H s in RAG2-complemented, G1-arrested parental ( n =4) and IAA-treated CTCF-degron ( n =6) v-Abl cells. Zoom-in profiles of indicated assays (mean ± s.e.m) for ±10kb regions of indicated V H s in G1-arrested parental ( n =3 for each assay) and IAA-treated CTCF-degron ( n =3 for each assay) lines along with putative CBE motif and orientation are presented. High-level signals for some V H s are off-scale on the y -axis for viewing low-level V H signals. See , for on-scale profiles. Other details are in and . p values were calculated using unpaired two-tailed t -test. V H s with decreased utilization upon substantial CTCF depletion are marked in blue. Detailed data for all V H s are shown in .

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Average signal counts ± s.e.m. of GRO-seq from three biologically independent experiments across 3’ part of Igh locus as diagramed at the top in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. S/AS: sense/anti-sense transcription. b, Average 3C-HTGTS signal counts ± s.e.m. from three independent experiments with RC-based baits (red asterisk) across 3’ part of Igh locus as diagramed at the top in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. c, Scatter plots of transcriptome-wide GRO-seq counts in G1-arrested parental ( x axis) and IAA-treated CTCF-degron ( y axis) cells. Three biologically independent repeats with similar results. Representative known requisite genes for V(D)J recombination and chromatin interaction are highlighted by red circles and blue arrows. Spearman’s correlation coefficient (r) and p values determined by two-sided Spearman’s correlation test are presented. d, Scatter plots of normalized GRO-seq read counts ( x axis) and 3C-HTGTS junction counts ( y axis) calculated from regions within ±5kb of highly-utilized V H s across four indicated V H domains in IAA-treated CTCF-degron cells. Data were normalized and quantified among three biologically independent experiments for each assay. Spearman’s correlation coefficient (r) and p values determined by two-sided Spearman’s correlation test are presented. See Methods for details.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques:

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, b, Average signal counts ± s.e.m. of CTCF ( a ) and Rad21 ( b ) ChIP-seq from three biologically independent experiments across 3’ part of Igh locus as diagramed at the top in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. “<”/”>”: orientation of indicated CBEs. c, d, CTCF ( c ) and Rad21 ( d ) ChIP-seq signal across all peaks genome-wide called in G1-arrested parental cells. See legend for other details. Three biologically independent repeats with similar results. IAA-treated CTCF-degron cells had greatly diminished CTCF and Rad21 chromatin occupancy, but, also had residual binding at some CBEs genome-wide. See Methods for details. Notably, CTCF binding increased at the RC and at a subset transcribed V H s ( , ) upon CTCF depletion. While increases in apparent CTCF-binding to the non-CBE-containing RC and also transcribed V H s upon CTCF depletion could be considered surprising, it may likely occur indirectly due to cohesin-mediated loop extrusion-driven dynamic associations of the RC dynamic anchor with residual-CTCF-bound CBEs across Igh.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: ChIP-sequencing, Genome Wide, Binding Assay

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a-h, HTGTS-V(D)J-seq analysis showing average utilization frequencies ± s.d. of V H segments across four highlighted V H domains in RAG2-complemented and G1-arrested parental ( a , b ), OsTir1-expressing ( , d ), CTCF-tagged ( e , f ), and CTCF-degron ( g , h ) cells without or with IAA treatment. n =4 ( a - f ) or n =6 ( g , h ) biologically independent repeats with similar results. Utilization of V H s presented in b - h are compared to those in NT parental cells shown in a , with red, blue, and grey bars indicating V H s with significantly increased, decreased, and unchanged utilization, respectively. To facilitate direct comparisons, the HTGTS-V(D)J-seq data for the IAA-treated parental and CTCF-degron cells shown in b and h are the same as those presented in and , respectively, with different y -axis scales. p values were calculated using unpaired two-tailed t -test (*: p ≤ 0.05, **: p ≤ 0.01, and ***: p ≤ 0.001). See for exact p values. See Methods for details.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Expressing, Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Schematic of the entire murine Igh locus with details as shown in a . b, The four V H domains are highlighted different colors which are maintained in related figures. c-e, Average signal counts ± s.e.m. of CTCF ( c ) and Rad21 ( d ) ChIP-seq and GRO-seq ( e ) from three biologically independent experiments across the four highlighted V H domains in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. S/AS: sense/anti-sense transcription. See , and for the plots across other parts of Igh locus. V H s for comparisons in are highlighted. f-i, Box-and-Whisker plots are presented with median, upper and lower quartiles and whiskers showing 1.5x interquartile range for CTCF ( f ) and Rad21 ( g ) ChIP-seq, GRO-seq ( h ), and 3C-HTGTS ( i ) signal counts within indicated V H domains in G1-arrested parental (“Par.”) and IAA-treated CTCF-degron cells. n =3 biological repeats with similar results for each condition in f-i . p values were calculated using two-sided Mann-Whitney U test. See Methods for details. Comparison of panels ( f ) and ( h ) reveals that residual CTCF binding levels are significantly higher in the highly transcribed distal V H domain than those of the proximal domains with less overall transcription, which might reflect known roles of transcription in CTCF chromatin binding , .

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: ChIP-sequencing, Whisker Assay, MANN-WHITNEY, Comparison, Binding Assay