Capture efficiency of probes with different GC-content (percentage of GC bases from the total bases) for SA and MSA-Cap HapMap (NA12813) libraries.

<p>The efficiency is shown as median depth of coverage of probe regions with different GC percentage. The GC percentage range of the probes is shown on the x axis. The median depth of coverage for the probes in the HapMap sample, either SA or MSA-Cap libraries, is represented on the left y axis. The percentage of probes in each GC content is shown on the right y axis.</p

Copy number profiles estimated by deep sequencing of PCL-tumour samples, prepared using the SA- or MSA-Cap methods.

<p>Chromosomes 1 to 22 plus X and Y are shown on the x axis. The log₂ ratio of the normalized by the normal depth values for the PCL-tumour libraries is represented on the y axis. Arrows point towards the detected by deep sequencing and previously identified by SNP6 array copy number changes: gain (6p), del (6q), del (12p), del (13q) and del (17p).</p

Mutational spectrum from SA and MSA-Cap libraries, prepared from PCL-tumour or PCL-buccal swab samples.

<p>Non-filtered variants were identified in each sample using Broad Institute Best Practice pipeline version 1.0.5273 upon comparison to human hg19 reference sequence and categorized according to putative gene effect. The cumulative number of variants for each library type is shown on the x axis and the sample and library type on the y axis. Variant categories, as assigned by the Ensembl database, are on the right side of the figure.</p

Genotype concordance for SNP calls in a PCL-tumour sample and a HapMap (NA12813) sample.

<p>For the PCL-tumour sample, the variants called by deep sequencing were compared to data from Affymetrix SNP 6.0 DNA array while for the HapMap sample published data were used for the comparison.</p

Reversion analysis of <i>Parp1</i> mutants.

<p><b>A.</b> Schemes used for isolation of revertants from the D5 (intron one) <i>Parp1</i> mutant ES cells. <b>B.</b> Three FIAU-resistant clones isolated after transfection of clone D5 with PB transposase (Rev1–3, grey and black) have regained sensitivity to olaparib similar to wild type cells (green). <i>Brca2</i>-deficient ES cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061520#pone.0061520-Farmer1" target="_blank">[8]</a>, which are sensitive to PARP inhibition, are shown for comparison. Error bars show SEM, <i>n = </i>5. ***, <i>P</i><0.001; comparison shown for D5Rev1 and <i>Parp1</i> mutant. <b>C.</b> Dose response curve for BMN 673 for three clones isolated using the G418 selection scheme. <i>n = 5</i>; ***, <i>P</i><0.001; comparison shown for G418 R2 and <i>Parp1</i> mutant. <b>D.</b> The G418 resistant clone that remains PARP inhibitor resistant (clone R1) is still a <i>Parp1</i> mutant. Lysates from the indicated cells were probed with anti-PARP followed by anti-β-tubulin. <b>E.</b> Clone G418 R1 still contains the original <i>Parp1</i> insertion. DNA from the indicated cells (same order as above) was analysed for the presence of the transposon-genome junction by PCR using the primers shown in <b>A</b>. <b>F.</b> Most clones isolated from the screen have a diploid DNA content. Three out of nine tested had a mixture of haploid and diploid cells similar to D2 (top, this clone also has the intron 1 <i>Parp1</i> insertion); all others were fully diploid including clone D5 (bottom).</p

Transposon insertions in other PARP superfamily members identified in sequencing of insertion sites from H3L1–H3L8.

<p>Transposon insertions in other PARP superfamily members identified in sequencing of insertion sites from H3L1–H3L8.</p

Insertion sites mapped in olaparib-resistant mutants from libraries H3L2–H3L13.

<p>Insertion sites mapped in olaparib-resistant mutants from libraries H3L2–H3L13.</p

Generating and screening haploid transposon mutant libraries.

<p><b>A.</b> The piggyBac transposon used for mutagenesis. The transposon cargo contains splice acceptors that disrupt transcription, but gene trapping is not directly selected for. PuroΔTK, is a positive-negative selection marker: puromycin can be used to select for integrations, and FIAU to select for loss of the transposon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061520#pone.0061520-Chen1" target="_blank">[30]</a>. <b>B.</b> Outline of the mutagenesis and screening process. A detailed protocol is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061520#pone.0061520.s002" target="_blank">Protocol S1</a>. <b>C.</b> The mutant pool remains predominantly haploid, as shown by propidium iodide staining of fixed cells from library H3L1. <b>D.</b> Determining drug concentration for screening. Olaparib was used at a concentration that kills >2.5×10⁵ wild type haploid cells (4 µM). <b>E.</b> Scheme for further analysis of clones of interest by transposon reversion.</p

Flow chart comparing the standard Agilent Whole Exome Target Enrichment protocol (SA protocol, v. 2.0.1), and the Minimal Starting Amount Capture (MSA-Cap) protocol.

<p>Flow chart comparing the standard Agilent Whole Exome Target Enrichment protocol (SA protocol, v. 2.0.1), and the Minimal Starting Amount Capture (MSA-Cap) protocol.</p

Silencing of endogenous enhances oxytocin receptor signalling via p44/42 MAPK in Hs578T cells

<p><b>Copyright information:</b></p><p>Taken from "Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers"</p><p>http://breast-cancer-research.com/content/9/6/R85</p><p>Breast Cancer Research : BCR 2007;9(6):R85-R85.</p><p>Published online 8 Dec 2007</p><p>PMCID:PMC2246188.</p><p></p> Hs578T cells transfected with either a scrambled control small interfering (si)RNA (SiCON) or an siRNA targeting (siRGS) were serum and insulin starved for 2 hours then stimulated with 1 × 10mol/l oxytocin. Cells were harvested at timepoints up to 120 minutes after oxytocin addition and lysates analysed for levels of phospho- and total p44/42 mitogen-activated protein kinase (MAPK) by immunoblotting. Quantitation of siRNA knockdown from two independent transfections. The upper plot is the quantitation of the blots shown in panel a. siRGS2 caused a significant (< 0.001) increase in phosphorylation in response to oxytocin. Quantitative real-time PCR analysis of expression in Hs578T cells transfected with the siRGS2. Each bar represents the mean ± 95% confidence limits of the fold difference in expression compared with the mean expression in the siCON transfected cells. Data from four samples harvested from two independent transfections is shown (one of which was also used in the lower oxytocin response experiment shown in panel b). Significant differences are indicated (**< 0.01)