6430 Nucleic Acids Research, 2011, Vol. 39, No. 15 centrifugation at 14 000g for 25 min at 4 C and the super- natants were used for immunoprecipitations. For immunoprecipitation, 1–2 mg nuclear extracts were incubated with 5 ml anti-MSL2, 5 ml anti-MOF or no antibody for 45 min at 4 C, with agitation. The antibody complexes were precipitated by incubation with 75 ml of Dynabeads conjugated to protein-A (Invitrogen) for 30 min at 4 C, with agitation. The supernatant was removed and the beads were washed twice with PBS (150 mM NaCl), 0.1% Triton X-100, 32 U/ml RNasin, 0.5 Protease inhibitor cocktail and twice in PBS (300 mM NaCl), 0.1% Triton X-100, 32 U/ml RNasin, 0.5 Protease inhibitor cocktail. The cross-linking in FA samples was reversed by adding 200 ml 0.45 M LiCl to the beads and incubating for 3–4 h at 65 C. RNA was isolated using TRI-reagent (Ambion), followed by puriﬁcation using an RNeasy kit (Qiagen) according to the instruc- tions by the suppliers. The RNA samples were concentrated and reverse-transcribed into cDNA using random primers with an ImPromII ﬁrst-strand synthesis kit (Promega). The single-stranded cDNA was puriﬁed with a QIAquick PCR puriﬁcation Kit (Qiagen) then the puriﬁed cDNA samples were ampliﬁed using a WGA2 GenomePlex Complete whole genome ampliﬁcation kit (Sigma), according to the manufacturer’s recommendations. For tiling array analysis, ampliﬁed DNA samples were fragmented, labeled and hybridized to an Affymetrix Drosophila Genome 2.0 array. The signal intensity data were analyzed with the Affymetrix Tiling Analysis Software (v. 1.1.02) using 90 bp bandwidth and perfect match only. For absolute amount analysis (transcript proﬁle), the bandwidth was set to zero. RNA enrichment ratios for all genes in nuclear extract N6 were calculated as the average enrichment ratio value of all probes located within exons of each gene. Total amounts of nuclear gene transcript were calculated from the corresponding input sample as the average value of all probes located within exons of each gene. Only genes with at least 10 probes within exons were included. For the comparison of our RIP proﬁles to MSL-complex binding proﬁle the ChIP-chip data for MSL1, MSL3 and MOF from (32) were downloaded from the ArrayExpress database (acces- sion number M-EXP-1508). The signal intensity data were analyzed with the Affymetrix Tiling Analysis Software (v. 1.1.02) using parameters: 300 bp bandwidth and perfect match only. The data were converted to D. melanogaster genomic release 5 using the Coordinates Converter Tool at FlyBase (33). RIP-qPCR Three biological replicates of native nuclear preps from Schneider’s line 2 cells (ATCC CRL-1963) were prepared as described above. The nuclei were washed once in lysis buffer and fractionation into nucleoplasm and chromatin was done essentially as described in (34). The pelleted nuclei were resuspended in 2 ml nuclear lysis buffer (20 mM HEPES pH 7.9, 3 mM EDTA, 10% glycerol, 150 mM KAc, 1.5 mM MgCl2, 0.1% Triton X-100, 1 mM dithiothreitol, 0.5 mM PMSF, 20 U/ml RNasin and 1 Protease inhibitor cocktail) and centrifuged at 14 000g for 30 min at 4 C. The supernatant was collected as the nucleoplasmic fraction. The pelleted chromatin was resuspended in 2.8 ml of nuclear lysis buffer and solubilized by sonication for 6 min at high setting (30 s ON, 30 s OFF). Cellular debris was removed by centrifugation at 14 000g for 20 min at 4 C and the supernatants were collected as the chromatin fractions. For immunoprecipitation, 5–10 mg nucleoplasmic and corresponding cytoplasmic extracts, respectively, were incubated with 5 ml anti-MSL2 (replicate A) or 2 ml anti-MSL2 serum (replicate B and C) for 45 min at 4 C with agitation. The antibody complexes were then precipitated as described above. RNA was isolated using TRI-reagent (Ambion), followed by DNase treatment using TURBO DNA-free kit (Ambion) and puriﬁcation using an RNeasy kit (Qiagen) according to the manufac- turer’s instruction. For replicate C, poly(A)+ RNA was isolated using Dynabeads Oligo (dT)25 (Invitrogen) in line with the instruction by the suppliers. The RNA samples were reverse-transcribed into cDNA using iScript cDNA synthesis kit (Bio-Rad). The RIP cDNA and the corresponding total cDNA were quantiﬁed by real-time PCR using SYBR green supermix (Bio-Rad). The primer pairs used were: roX1 (50-TACCGCTCTCTTTCGGGACTTG-30, 50-TCCATC ACTCTCTATCGGGCTG-30), roX2 (50-GGCCATCGA AAGGGTAAATTG-30, 50-ACTGTCCGTAAGACAAT TCAAC-30), msl2 (50-CTGGACACGAATAGTGAA GCC-30, 50-TTGCAGCAATCCCAGCATC-30), actin (50-CAGCCAGCAGTCGTCTAATC-30, 50-ACAACCA GAGCAGCAACTTC-30) and Rpl32 (50-CGATGTTGG GCATCAGATAC-30, 50-CCCAAGATCGTGAAGAAG C-30). The relative enrichment levels were calculated in relation to actin cDNA in each replicate. RESULTS To determine the potential association of the MSL complex with RNA moieties in an unbiased procedure, we performed RIP experiments on nuclear extracts from S2 cells, with and without cross-linking, followed by tiling-array analysis (RIP-chip) (Figure 1). For the RIP experiment, we chose MSL2 as a representative of the MSL-complex scaffold (consisting of MSL1 and MSL2) required for the complex to bind DNA and MOF as a representative of the complex members required for spreading (1,35). MSL-complex associates with roX1 and roX2 RNAs from the X chromosome We ﬁrst investigated the enrichment of RNAs transcribed from the X chromosome in the MSL2- and MOF-RIPs. We found that, as predicted, roX1 and roX2 are highly enriched in the complex (Figure 1). We found no evidence of any additional X-linked non-coding RNA associated with the MSL complex when roX1 and roX2 are present (Figure 1). Neither did we ﬁnd any evidence that the MSL-complex attracts any cryptic RNA species produced at the MREs. The overall enrichment levels of