Additional file 1: Figure S1. of Predicting chromatin organization using histone marks

Prediction of Jin2013 hubs. (A) Distribution of chromatin anchors interactions frequency. Top 10 % are defined as hubs. (B) Percentage of super-enhancers covered by hubs. (C) Prediction accuracy using DNA sequence genetic features, including PhastCons conservation score, TSS proximity and GC content. AUC scores are shown in parentheses. (D) Prediction accuracy using individual histone marks. AUC scores are shown in parentheses. (E) Hubs prediction performance for hubs defined using different thresholds of interactions frequency. (F) Hubs prediction performance with various number of trees. Figure S2. Prediction of Rao2014 hubs. (A) Distribution of chromatin anchors interactions frequency. Top 10 % are defined as hubs. (B) Prediction accuracy using individual histone marks. AUC scores are shown in parentheses. Figure S3. TAD boundary prediction accuracy using individual histone marks. (PDF 1896 kb

Generation of tagged cyclin E1 knock-In mice and analyses of cyclin E1-containing protein complexes.

<p>(A and B) Targeting strategy to knock-in Flag and HA tags into <i>the cyclin E1</i> locus to generate N-terminally tagged <i>cyclin E1</i>^<i>Ntag</i> (A) and C-terminally tagged <i>cyclin E1</i>^<i>Ctag</i> alleles (B). The exons are shown as green boxes, Flag tag as a blue box, and HA tag as a red box. Start and stop codons are marked with orange and yellow arrowheads, respectively. The hygromycin resistance cassette (Hyg) with flanking loxP sequences (filled arrows) is also indicated. Restriction enzyme recognition sites: E, EcoRI; A, AflII; Sc, ScaI; N, NotI; X, XhoI; K, KpnI; S, SpeI; P, PmeI; Hp, HpaI. Note that panel (A) was shown in ref [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (C) Western blot analysis of wild-type control (Ctrl), heterozygous cyclin <i>E1</i>^{<i>+/Ntag</i>}, <i>cyclin E1</i>^{<i>+/Ctag</i>}, and <i>cyclin E1</i>^{<i>Ntag/Ctag</i>} embryonic stem cells probed with anti-cyclin E1 and -HA antibodies. Actin served as a loading control. Forth panel: cyclin E1 was immunoprecipitated with anti-Flag antibody and the immunoblots were probed with anti-Cdk2 antibody. Fifth panel: anti-Flag immunoprecipitates were used for <i>in vitro</i> kinase reactions using histone H1 as a substrate. (D) Same analyses as in (C) using spleens of homozygous knock-in mice. Lanes 1–2 in panels (C and D) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (E) Cyclin E levels detected by western blotting in the indicated organs of 1-month-old mice and in embryonic brain (day E14.5). Actin served as a loading control. The last two lanes (Brain) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (F) Quantification of cyclin E levels in different organs, normalized against actin (from E). (G) Protein lysates from brains and testes of adult tagged cyclin E1 knock-in mice were separated by gel-filtration chromatography. Fractions containing protein complexes of the indicated molecular weights were analyzed by western blotting for cyclin E using an anti-HA antibody. (H) Cyclin E1-associated proteins were purified from the indicated organs of tagged cyclin E1 knock-in (KI) mice, or from control mice (Ctrl, ‘mock’ purifications) by sequential immunoaffinity purifications with anti-Flag and -HA antibodies, and 10% of the final eluate was resolved on PAGE gels and silver-stained. Arrows indicate bands corresponding to cyclin E1. Panels representing embryonic and adult brains were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>].</p

Additional file 8: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S7. UDiTaS characterization of plasmid standards without carrier DNA. To ensure that the carrier mouse genomic DNA was not influencing the UDiTaS reaction, additional sets of UDiTaS reactions were run with plasmids in the absence of any carrier DNA. a. CEP290 plasmids with the Wild Type, Large Deletion, and Large Insertion (PLA379, PLA367, and PLA370) and b. B2M-TRAC plasmids with the B2M, TRAC, and both balanced translocations (PLA377, PLA378, PLA365, and PLA366) were diluted as described in the methods. The DNA plasmids mixtures were process through UDiTaS and the analysis pipeline. Plotted is the expected frequency for a given structural variant vs. measured frequency for a structural variant (x = y is the grey line). Accuracy and linearity appear to be excellent for both loci with all four primers, with an LLOD of ~ 0.01%-0.1%. (PPTX 991 kb

Quantitative proteomic (iTRAQ) analysis of cyclin E1-interacting proteins in mouse organs in the absence of Cdk2.

<p>(A) Relative abundance of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of <i>Cdk2</i>^<i>-/-</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} mice, as compared to <i>Cdk2</i>^<i>+/+</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} animals, was determined by iTRAQ labeling and LC-MS. (B) The amount of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of wild-type (Ctrl), <i>Cdk2</i>^<i>+/+</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} (KI), and <i>Cdk2</i>^<i>-/-</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} (Cdk2^<i>-/-</i>) mice was gauged by immunoprecipitation with an anti-Flag antibody and immunoblotting with the indicated antibodies. Abundance of each protein in total lysates (whole) is also shown. (C) Spleens from wild-type mice were incubated with 20 μM CVT-313 (+) or with vehicle only (-). Association of cyclin E1 with Cdk2, Cdk1, Cdk4 and Cdk5 was assessed by IP–western blotting. Whole, whole cell lysate from vehicle only-treated mice. Lower panel: To ensure that CVT-313 treatment inhibited Cdk2 kinase activity, Cdk2 was immunoprecipitated from lysates and used for <i>in vitro</i> kinase reactions with histone H1 as a substrate. Note that CVT-313 treatment strongly decreased Cdk2 kinase activity.</p

Additional file 1: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S1. Schematic of the bioinformatics pipeline for UDiTaS analysis. (PPTX 61 kb

Cyclin E1-interactomes.

<p>(A) Diagrams depicting cyclin E1-interacting proteins in the indicated mouse organs. Cyclin E1 is shown as a red node. Green nodes denote highest-confidence ‘core’ interactors (Category 1, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Yellow and blue nodes represent, respectively, lower confidence Categories 2 and 3 interactors that were included to the interactome based on their reported interaction with core interactors in the STRING database (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Solid lines depict STRING-verified interactions. Dashed lines depict an interaction derived from our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other core interactors. (B) A combined diagram depicting cyclin E1-interacting proteins from all five organs analyzed. Cyclin E1 is shown as a red node. Green nodes denote highest-confidence core (Category 1) interactors. Yellow and blue nodes denote, respectively, Categories 2 and 3 interactors, which were included into the interactome based on their ability to interact with core interactors as revealed by STRING (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Solid blue lines depict STRING-verified interactions between pairs of proteins that were identified by us as cyclin E1-interacting proteins within the same organ. Gray dotted lines depict STRING-verified interactions between pairs of proteins identified as cyclin E1-interators in different organs. Blue dashed lines depict interactions detected in our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other core proteins within the same organ interactome.</p

Analyses of cyclin E1-interactomes.

<p>(A) Venn diagram depicting the numbers of unique and shared cyclin E1 interactors in the indicated organs. (B) Fraction of unique interactors in the indicated organs. (C) Pairwise comparisons of the fraction of cyclin E1-interactors shared between the indicated organs. (D) The fraction of cyclin E1 interactors in the indicated organs that were assigned to a given Gene Ontology category (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s008" target="_blank">S2 Table</a>). Categories assigned at least 10% of the interactors in a given organ are marked in red. CC, cell cycle; TX, transcription; Neuro, neuronal function; Cyto, microtubules/cytoskeleton; Ubiq, ubiquitination; Metab, metabolism. (E) Heatmap displaying functional enrichment of cyclin E1 interactors in Gene Ontology classes of biological processes. The five columns correspond to the five organs analyzed, and each horizontal row denotes a distinct biological process. Colors depict fold-enrichment for the cyclin E1 interactors from the particular organ in a given biological process, between green (fold-enrichment one or lower) to red (fold-enrichment five or higher). Only categories in which at least one organ had an EASE score of 0.2 or lower are shown (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Left panel shows a complete heatmap, right panels show selected common and organ-specific biological processes: cell cycle (red box, enriched in all organs), neurogenesis and synaptic plasticity (blue box, shared between embryonic and adult brains) and regulation of microtubule-based processes and microtubule cytoskeleton (green box, specific to adult brain).</p

Additional file 6: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S5. Genome mapping rates for UDiTaS. Individual reads map to the expected genome site with high frequency indicating the robustness of the assay. Ten distinct samples for primer OLI6062 are plotted on the x-axis and the y-axis shows the percentage or reads mapping to the expected reference amplicon for each sample. (PPTX 3767 kb

Predicted Cdk2 substrates in the organ-specific cyclin E1-interactome.

<p>The fraction of predicted Cdk substrates among interactors of cyclin E1 in the indicated organs. The lists of interactors in each organ were analyzed with Scansite 3.0, using high-stringency or medium-stringency scoring.</p

Additional file 7: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S6. UDiTaS characterization and comparison to AMP-Seq with plasmid standards. Plasmids containing the CEP290 structural variants a. or the TRAC-B2M balanced translocation b. and c. were synthesized and contain engineered unique SNPs in the insert to identify the plasmid after sequencing. The plasmids were diluted at various levels into mouse genomic DNA and processed through UDiTaS and AMP-Seq using primers for CEP290 a., B2M b. and TRAC c. The number of input plasmids versus the number of plasmids detected is plotted for both UDiTaS and AMP-Seq. Linear regression models and 95% confidence model predictions are displayed on the plots. The parameter β determines the linearity of the method, with values close to 1 indicating more linearity. We used ANOVA p-values to examine differences in β for UDiTaS and AMP-Seq. Below each plot, the table displays the total number of fastq reads sequenced in the reaction, the number of reads mapped to the wild-type amplicon (the most abundant one) and the final number of UMIs counted, for both UDiTaS and AMP-Seq. At all tested loci, UDiTaS shows greater linearity and number of UMIs detected when compared to AMP-Seq. (PPTX 12722 kb