1. Transcription within a Functional Human Centromere
Richard Saffery, Huseyin Sumer, Sara Hassan, Lee H. Wong, Jeffrey M. Craig, Kazuo Todokoro, Melissa Anderson, Angela Stafford, K.H.Andy Choo 
Molecular Cell 
Volume 12, Issue 2, Pages (August 2003)
DOI: /S (03)00279-X

2. Figure 1 NC Gene Expression and Domain Organization
(A) BAC array spanning a total of 8 Mb, showing positions of clones (horizontal bars) used in ChIP- and S/MAR-array analyses. Positions and orientations of genes located at 10q25 used in expression study are shown by arrows or arrowheads (green for expressed genes, red for nonexpressed genes).
(B) Scaffold/matrix attachment along the 10q25 BAC array as determined by S/MAR-array analysis on chromatin prepared from mardel(10) and normal chromosome 10-containing hybrid cell lines, denoted as M10 and N10, respectively. Data points, represented on the x axis by the midpoints of the positions of the BACs relative to the start of the contig map, were expressed as the means and standard deviation of the means from ten independent experiments, and were calculated as the percentage difference between the scaffold/matrix-attached/unattached signal ratio of M10 and N10 (y axis). Significance of the data points was determined using a Student's t test and is indicated by *p < A summary of S/MAR-FISH data is shown at the top of the graph, denoting scaffold/matrix-attached (+) or nonattached (−) BAC DNA, and showing close concordance with the S/MAR-array results. Corresponding BACs on the graph used in FISH are shown as open circles. The S/MAR- and CENP-A-associated (Lo et al., 2001a) domains are indicated by blue and purple shadings, respectively.
(C) Distribution of CENP-H protein along the 10q25 BAC contig (x axis) as determined by ChIP-array analysis. The y axis shows the fold difference between the normalized bound/input ratio of M10 and N10 cell lines. Each data point is the mean of four independent experiments. Significance of the data points was determined using a Student's t test and is indicated by *p < Green shading indicates position of the CENP-H-associated region.
(D) Distribution of HP1 protein along the 10q25 BAC contig (x axis) as determined by ChIP-array analysis. The y axis shows the fold difference between the normalized bound/input ratio of M10 and N10 cell lines. Each data point is the mean of four independent experiments. Significance (*p < 0.01) of the data points was determined using a Student's t test. Orange shading indicates the position of the HP1-associated domain.
Molecular Cell  , DOI: ( /S (03)00279-X)

3. Figure 2 CT Analysis of Expressed Genes
Refer to Table 1 for explanation of CT and ΔCT. 1/ΔCT (y axis) provides a measure of expression level of individual genes.
(A) Comparison of SYBR green results for somatic cell hybrids containing human chromosome 10 (shown as gray bars) and those for hybrids containing mardel(10) (black bars) indicate no major difference between hybrid pairs for all genes tested. See Table 1 for a summary of the relative expression levels between hybrids and Student's t test values.
(B) SYBR green-based real-time RT-PCR analysis of a mardel(10)-containing mouse ES hybrid cell line (ES-M10) showing CT values of expressed genes including hCG39837 (spanning the CENP-A-associated domain).
(C) Gel electrophoresis of RT-PCR products obtained by SYBR green and TAQman analyses of hCG39837, showing specificity of gene amplification. ES-WT refers to wild-type ES cells, from which monochromosomal mardel(10)-containing hybrid lines ES-M10-9, -19, and -20 were derived. NT, no template control; S, results from SYBR green; and T, results from TAQman experiments.
Molecular Cell  , DOI: ( /S (03)00279-X)

4. Figure 3
Visualization of Scaffold/Matrix Attachment on Metaphase Chromosomes
FISH on histone-depleted chromosomes was performed as published (Bickmore and Oghene, 1996).
(A) Chromosome-10 centromeric α satellite probe, pα3.5 (Devilee et al., 1988), showing tightly packed signals (closed arrow).
(B, E, F, and I) BAC clones bA313D6 (B and F) and bA427L15 (E and I), which map outside the S/MAR-enriched domain identified by S/MAR-array analysis, produce dispersed signals (open arrows) on both the normal chromosome 10 (left panel) and mardel(10) (right panel), indicating predominantly non-scaffold/matrix attachment of the probed regions.
(C, D, G, and H) BAC clones E8 (C and G) and bA153G5 (D and H), which map within the S/MAR-enriched domain identified by S/MAR-array analysis, produce dispersed signals (open arrow) on chromosome 10 (C and D) but tightly packed signals on mardel(10) (closed arrow; [G] and [H]), confirming increased scaffold/matrix attachment for these regions on mardel(10). Combined image (i) and split images for DAPI (ii) and FITC (iii) are shown for each probe. Scale bar represents 1 μM.
Molecular Cell  , DOI: ( /S (03)00279-X)

5. Figure 4 Summary of Domain Distribution at the 10q25 Neocentromere
Organization of various modified chromatin domains is shown in relation to the expression status of underlying genes 1–15 (Figure 1A). “+” denotes positive gene expression following NC formation, whereas “?” indicates unknown expression status of a gene present in the HP1-associated chromatin domain.
Molecular Cell  , DOI: ( /S (03)00279-X)