!Citation=Bing H, et al. Genome Research. 2012. Submitted. !Title=Mapping the Centromeric Heterochromatin by Comparative Genomic Hybridization Analysis and Chromosome Deletions in Drosophila Melanogaster !PubMedID= !Name=Array CGH of Drosophila compound chromosomes on heterochromatin custom array !ExptSetNo=6721 !Description=CGH analysis of embryos lacking specific chromosomes or chromosome arms. For each experiment 100-150 embryos of the appropriate genotype were collected, dechorionated and digested with proteinase K prior to phenol/chloroform extraction. DNA was then sonicated and sodium acetate/ethanol precipitated. DNA extracted from randomly staged 0-8 hr wild type Oregon R embryos was used as reference for all experiments. Purified DNA was labeled using the BioPrime kit (Life Technologies) and hybridized with the microarray following standard Agilent CGH protocol. Feature extraction was performed by Agilent feature extraction software using the CGH protocol. Embryos with no X chromosome were obtained by crossing attached-X/Y females to X/Y males. The stock used was C(1)DX, y f (Wieschaus and Sweeton, 1988). Embryos with no X and Y chromosomes were obtained by crossing attached-X/Y females (C(1)RM, y2suwawa) to attached-XY males (YSX YL, In(1)EN, y B). The compound II chromosomes RM(2L); RM(2R)=C(2)v and the compound III chromosomes RM(3L); RM(3R)=C(3)se, in which the two left arms or the two right arms segregate together, were used to generate 2L- and 2R-, and 3L- and 3R- embryos, respectively(Merrill et al., 1988). The compound II C(2)EN and compound III C(3)EN st1 cu1es, stocks (Bloomington 2974 and 1117) were used to generate embryos deficient for the entire second and third chromosome, respectively. The compound IV C(4)RM, ci1eyR/0 (Bloomington 1785) were used to generate embryos deficient for the fourth chromosome. Embryos deficient for chromosome 4 were identified by their defects in denticle belt patterning during late embryogenesis, whereas embryos deficient for other chromosome/chromosome arm were recognized based on their specific phenotypic defects during early embryonic development (Wieschaus and Sweeton, 1988; Merrill et al, 1988) . The early embryonic development phenotypes can be easily recognized by covering embryos in oil to make them transparent, and then viewing them in transmitted light under a stereomicroscope. All embryos were collected at room temperature. !Name=Array CGH of Drosophila translocations on heterochromatin custom array-eu/het boundary !ExptSetNo=6722 !Description=CGH analysis of translocations with breakpoints at the euchromatin/heterochromatin boundary of 2L, 3L and X. To obtain embryos lacking the euchromatin portion of the chromosome arms, translocation males bearing breakpoint at the euchromatin/heterochromatin boundary of 2L, 3L and X (Lindslet et al, 1972) were crossed to C(2)EN, C(3)EN or attached X females, respectively. !Name=Array CGH of Drosophila ChrX deficiencies/duplications on heterochromatin custom array !ExptSetNo=6723 !Description=168 Dp(1;Y)y[2]67g 175 Dp(1;Y)y[2]sc 114n Dp(1;Y)y[+]mal[126] 114f Df(1)fog[114] 186n Dp(1;Y)y[+]mal 186f Df(1)mal12 B2580 Dp(1;Y)ct y[+] B5280 Dp(1;Y)ct[+]y[+] CGH analysis of the X chromosome rearrangements, including (1) Y duplicated for a piece of proximal X and (2) X deficiencies encompassing part or all of the X heterochromatin. To generate embryos lacking different portions of the X heterochromatin, two types of stocks were used: (1) males carrying a deficient X chromosome missing part of the heterochromatin (generally fog-) and a duplication on the Y chromosome that complements the deficiency, and (2) males carrying a duplication of X on the Y chromosome which covers part of the X heterochromatin were crossed to attached X females. In both cases males were crossed to attached X females. In the first case, one quarter of embryos lack most of the X chromosome except the duplication of X on Y. These embryos were identified according to their defects during cellularization. Another quarter of the embryos carry the deficient X chromosome as their sole X chromosome. These embryos were identified according to their defects in posterior midgut formation during early gastrulation. In the second case, one quarter of embryos that lack most of the X chromosome except the duplication of X on Y were identified according to their defects in cellularization or posterior midgut formation. !Name=Array CGH of Drosophila translocations on heterochromatin custom array-2Rhet !ExptSetNo=6724 !Description=2219 T(2;3)ftz[Rpl] 130058 T(Y;2)B[SV5] 130093 T(Y;2)B63 130112 T(Y;2)B238 101579 T(2;3)bxd[68] 101988 T(Y;2)G10 107387 T(2;3)E(da) CGH analysis of translocations with breakpoints in 2R heterochromatin. To generate embryos deficient for smaller regions of 2R heterochromatin, T(2;Y) or T(2;3) translocation males were crossed to C(2)EN females. One eighth of the embryos that lack 2R were identified according to their failure in forming the ventral furrow during early gastrulation. !Name=Array CGH of Drosophila polytene chromosomes on heterochromatin custom array !ExptSetNo=6725 !Description=CGH analysis of Drosophila polytene chromosomes isolated from whole salivary glands dissected from roaming third-instar larvae or from ovaries dissected from adult females. Salivary gland (I) and (II) are two biological repeats. !Name=Gene expression analysis for Oregon R embryos at different developmental stages !ExptSetNo=6726 !Description=cDNA were prepared from manually staged wild type Oregon R embryos at various developmental stages (0-1 hr, 2-2.5 hr/cycle 13-early cycle 14, 2.5-3 hr/mid- to late cycle 14, 3-4 hr, 4-5 hr, 19-22 hr) and analyzed by microarray. cDNA prepared from Oregon R embryos with mixed developmental stages (0-16 hrs) were used as reference for hybridization. (I) and (II) are biological repeats. Oregon R embryos visually staged (0-1 hr, cycle13 to early cycle 14 (~2-2.5 hr), mid- to late cycle 14 (~2.5-3 hr), 3-4 hr, 4-5 hr and 19-22 hr) under a compound microscope were collected from apple juice-agar plates, dechorionated for 2 min in sodium hypochlorite (bleach), washed in water, and then frozen in 1 ml of heptane (Sigma) using a dry ice/ethanol chamber. Total RNA was extracted with TRIzol (Invitrogen), and 325 ng of RNA (approximately 5-10 embryos) was used to synthesize complementary RNA (cRNA) according to the Agilent protocol. cRNA prepared from an Oregon R embryo sample with broad developmental stages (0-16 hrs) was used as the reference for hybridization. Same amount of Cy5-labeled sample cRNA and Cy3-labeled reference cRNA were mixed together for hybridization such that there was at least 5 pmol of dye in each channel with equal quantity of cDNA (approximately 1000 ng of cRNA probes for each channel). Each array was hybridized with probes for 17 h in a 65 C oven rotating at 20 rpm. Arrays were washed according to the Agilent CGH protocol (including a 37?C wash) and subsequently scanned using Agilent scanner. Feature extraction was performed by Agilent feature extraction software using the gene expression protocol.