Atomic Force Microscopy Characterization of Palmitoylceramide and Cholesterol Effects on Phospholipid Bilayers: A Topographic and Nanomechanical Study

Supported planar bilayers (SPBs) on mica substrates have been studied at 23 °C under atomic force microscopy (AFM)-based surface topography and force spectroscopy with two main objectives: (i) to characterize palmitoylceramide (pCer)-induced gel (L_β) domains in binary mixtures with either its sphingolipid relative palmitoylsphingomyelin (pSM) or the glycerophospholipid dipalmitoylphosphorylcholine (DPPC) and (ii) to evaluate effects of incorporating cholesterol (Chol) into the previous mixtures in terms of Cer and Chol cooperation for the generation of lamellar gel (L_β) phases of ternary composition. Binary phospholipid/pCer mixtures at <i>X</i>_pCer < 0.33 promote the generation of laterally segregated micron-sized pCer-rich domains. Their analysis at different phospholipid/pCer ratios, by means of domain thickness, roughness, and mechanical resistance to tip piercing, reveals unvarying AFM-derived features over increasing pCer concentrations. These results suggest that the domains grow in size with increasing pCer concentrations while keeping a constant phospholipid/pCer stoichiometry. Moreover, the data show important differences between pCer interactions with pSM or DPPC. Gel domains generated in pSM/pCer bilayers are thinner than the pSM-rich surrounding phase, while the opposite is observed in DPPC/pCer mixtures. Furthermore, a higher breakthrough force is observed for pSM/pCer as compared to DPPC/pCer domains, which can be associated with the preferential pCer interaction with its sphingolipid relative pSM. Cholesterol incorporation into both binary mixtures at a high Chol and pCer ratio abolishes any phospholipid/pCer binary domains. Bilayers with properties different from any of the pure or binary samples are observed instead. The data support no displacement of Chol by pCer or vice versa under these conditions, but rather a preferential interaction between the two hydrophobic lipids

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection

<div><p>Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.</p></div

MOESM1 of “Same difference”: comprehensive evaluation of four DNA methylation measurement platforms

Additional file 1. Table S1. Library input details. Table S2. Target region properties and CpGs covered. Table S3. Sequencing details. Figure S1. Number of CpG-units covered, Mean and median coverage per CpG-unit. Figure S2. Intra- and Inter-platform CpG-unit overlap and methylation levels concordance. Table S4. Intra- and Inter-platform details. Figure S3. Overlap of exon annotation of CpG-units as UpSet plot. Figure S4. Overlap of intron annotation of CpG-units as UpSet plot. Figure S5. Overlap of promoters annotation of CpG-units as UpSet plot. Figure S6. Overlap of CpG island annotation of CpG-units as UpSet plot. Figure S7. Overlap CpG shores annotation of CpG-units as UpSet plot. Figure S8. Overlap of unannotated CpG-units as UpSet plot

Overlap of detected peaks and gene annotation overlap across multiplex levels.

<p>A) The percent overlap of peaks for each sample to 1-plex across multiplex levels. As multiplexing increases there is a shift towards decreasing overlap. However, the overlap of peaks called from multiplexed samples with peaks identified in the non-multiplexed sample is generally above 92%. B) The percent overlap of each sample pairwise across multiplexed libraries. Overlap among peaks called from samples with similar coverage is generally above 94%. There is a similar trend, as in A, that as multiplexing increases overlaps start to decrease. C) The overlap of gene annotations of peaks called from each multiplexed sample compared to peaks from the non-multiplexed sample. Generally, as multiplexing increases more than 90% of the genes annotated in 1-plex are present in the gene annotations of multiplexed libraries. D) The percent of overlap of gene annotations pairwise for each sample.</p

Peak characteristics.

<p>A) P-values for detected peaks shift towards reduced significance as multiplexing increases. B) The difference in peak apex position of peaks detected in multiplexed libraries to peak apex positions of peaks detected in the non-multiplexed library shows consistent difference across all multiplexed levels while increasing variability as multiplexing increases. C) Peak width distributions show a marginal reduction across multiplex levels.</p

Peak detection and false discovery rate.

<p>As expected the number of peaks and their width are reduced as coverage is reduced. A) The mean number of peaks identified for each sample by multiplex level. B) The mean peak width of identified peaks for each sample by multiplex level. C) The false discovery rate (FDR) for each sample was computed by contrasting the input to the IP samples: ~181 million (M) reads (blue), ~43M reads (green), ~43M reads chip-2x input (red), ~31M reads (orange), ~21M reads (salmon). FDR for some experiments related to Input-4 are close to >0.45% suggesting these rates are an artifact of the library. Overall, experiments either 1-plex or multiplexed, with equal fractions of IP and input or double input, the FDR is < = 0.43%.</p

ChIP-seq multiplexing sequencing scheme.

<p>The ChIP-seq multiplexing titration scheme consists of: one whole lane of ChIP sample (1-plex), one whole lane of input sample (1-plex), two lanes with 4 samples (4-plex) of 2 ChIP and 2 input samples in each lane, one lane with 6 samples (6-plex) of 1 input and 5 ChIP samples, and one lane with 8 samples (8-plex) of 1 input and 7 ChIP samples. Sample labels correspond to sample type and llumina TruSeq indexed used (e.g. ChIP-5 is IP library with index number 5)</p

Mapped reads summary.

<p>A) Unique mapped reads are considered to be the fraction of mapped reads after duplicate reads are removed. Multiplexed libraries yield proportionally more unique mapped reads per lane. B) Genome view of sequence coverage along the HoxA region (chr7: 27,132,000–27,139,000) showing consistent coverage across all multiplex levels with decreasing coverage as multiplexing increases.</p

Genomic microarray analysis of CENP-C and CENP-H binding domains in two independent 13q32/33 neocentromeres

Ideogrammatic representation of the two neocentric chromosomes analyzed. From left to right: a normal chromosome 13, the invdup13q21 in IMS13q with a neocentromere in band 13q32, and the invdup13q21 in BBB with a neocentromere in band 13q33.1. An expansion of the 13q31.3 to 13q33.2 area included in the bacterial artificial chromosome (BAC) CHIP is shown. The position and size of each previously mapped centromere protein (CENP)-A domain from Alonso and coworkers [32] are indicated. DNA obtained from chromatin immunoprecipitation (ChIP) using antibodies to CENP-C (circles) and CENP-H (triangles) from cell lines BBB and IMS13q was hybridized to a contiguous BAC microarray spanning 14 megabases (Mb) from 13q31.3 to 13q33.2. Shown across the bottom of the graph is the tiling path of the unique sequenced regions for each BAC, the previously determined CENP-A domains [32] in cell lines BBB and IMS13q, and the genes in the region. Three independent biologic replicates were performed for each ChIP from each cell line, and the scale normalized mean logCy-5:Cy-3 intensity ratios (ChIP to input) with standard error (SE) were plotted on the y-axis for each BAC. Positive intensity ratios were identified as those that were at least three times the standard deviation (SD) from the experimental mean (gray or black dashed lines; see Materials and methods). For cell line BBB, CENP-C ChIP, the experimental mean was 0 ± 0.82 SD. Positive values ≥ 2.5 (black dashed line) were as follows: alpha sat = 6.42 ± 0.39 SE and BAC RP11-46I10 = 4.66 ± 0.92 SE. BAC RP11-29B2 was slightly increased (1.18 ± 1.2 SE) but not statistically significantly. All other BACs ranged from -1.1 to ≤ 0.96. For cell line BBB, CENP-H ChIP, the experimental mean was -0.02 ± 0.75 SD. Positive values ≥ 2.2 (grey dashed line) were as follows: alpha sat = 4.92 ± 1.86 SE and BAC RP11-46I10 = 5.57 ± 0.77 SE. BAC RP11-29B2 was slightly increased (1.58 ± 0.71 SE) but not statistically significantly. All other BACs ranged from -1.27 to ≤ 1.03. For cell line IMS13q, CENP-C ChIP, the experimental mean was 0 ± 0.84 SD. Positive values ≥ 2.5 (black dashed line) were as follows: alpha sat = 5.26 ± 0.38 SE and BAC RP11-199B17 = 4.95 ± 0.86 SE. All other BACs ranged from -1.7 to ≤ 0.93. For cell line IMS13q, CENP-H, the experimental mean was 0.00 ± 0.64 SD. Positive values ≥ 1.9 (grey dashed line) were as follows: alpha sat = 2.63 ± 1.03 SE and BAC RP11-199B17 = 3.95 ± 1.06 SE. All other BACs ranged from -1.17 to ≤ 1.13. Expansion of BAC map in regions that are positive for CENP-C and CENP-H in each neocentromere examined, showing BAC names and overlaps, the genes, and the previously determined CENP-A domains. For cell line BBB, the CENP-A, CENP-C, and CENP-H were mapped to the identical BACs (negative for RP11-811P12, strongly positive for BAC 46I10, and weakly positive for 29B2). For cell line IMS13q, the CENP-A mapped to two contiguous BACs (RP11-721F4 and RP11-199B17), whereas CENP-C and CENP-H mapped only to one BAC (RP11-199B17).<p><b>Copyright information:</b></p><p>Taken from "Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres"</p><p>http://genomebiology.com/2007/8/7/R148</p><p>Genome Biology 2007;8(7):R148-R148.</p><p>Published online 25 Jul 2007</p><p>PMCID:PMC2323242.</p><p></p

The BBB neocentromere contains a major and a minor centromere chromatin domain

DNA obtained from chromatin immunoprecipitation (ChIP) using antibodies to CENP-A, CENP-C, and CENP-H from cell line BBB was hybridized to a custom made microarray containing 257 unique polymerase chain reaction (PCR) fragments. Three independent biological replicates were performed for each antibody, and the scale normalized mean logCy-5:Cy-3 intensity ratios (ChIP to input), were plotted on the y-axis with the standard error (SE) for each PCR fragment. Intensity ratios at least three times the standard deviation (SD) from the background mean (dashed line) were considered positives (see Materials and methods). An alpha satellite containing plasmid was included as a positive control (far right). Centromere protein (CENP)-A ChIP. The major CENP-A domain was about 80.3 kilobases (kb; shaded region), with positive intensity ratios 1.17 to 2.46. The minor domain was about 8.5 kb (shaded region) and was approximately 162 kb downstream from the major domain; intensity ratios were 1.14 to 1.33. Background experimental mean was -0.39 ± 0.47 SD, one-tailed distribution cut-off was ≤ 0.68, positive values were ≥ 1.02 (dashed line). Alpha satellite = 1.63 ± 0.18 SE. CENP-C ChIP. Major CENP-C domain was 87.8 kb (shaded region). Intensity ratios were 0.67 to 3.41. Minor domain was 8.5 kb; intensity ratios were 0.65 to 1.07 (shaded region). Background experimental mean was -0.37 ± 0.34 SD, one-tailed distribution cut-off was ≤ 0.31, positive values were ≥ 0.65 (dashed line). Alpha satellite = 2.36 ± 0.70 SE. CENP-H ChIP. Major CENP-H domain was about 86.3 kb (shaded region), and positive intensity ratios were 0.64 to 3.35. Minor domain was about 1.9 kb (shaded region), and intensity ratios were 0.82 and 1.14. Background experimental mean was -0.33 ± 0.32 SD, one-tailed distribution cutoff was ≤ 0.56, positive values were ≥ 0.63 (dashed lines). Alpha sat = 2.06 ± 0.59 SE. The 2.3 megabase (Mb) region included in the PCR CHIP. The central 350 kb region, covered by PCR fragments at high density. The adjacent megabase on either side of the central region, shown at a 10 fold reduced scale, was covered by PCR fragments at decreasing density. PCR microarray fragments listed in Table 1, found at the edges of CENP-A, CENP-C and CENP-H domains, and the negative values within the first domain, are shown. The major and minor chromatin domains are shown by the rectangles. The tiling path of the unique sequenced regions of each bacterial artificial chromosome (BAC) and their overlaps are shown within the 350 kb region. The corresponding Repeat Masker data from the Human Genome Browser at UCSC and thegenes in the area are indicated [50].<p><b>Copyright information:</b></p><p>Taken from "Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres"</p><p>http://genomebiology.com/2007/8/7/R148</p><p>Genome Biology 2007;8(7):R148-R148.</p><p>Published online 25 Jul 2007</p><p>PMCID:PMC2323242.</p><p></p