Nucleic Acids Research, 2011, Vol. 39, No. 15 6431 Figure 1. MSL-complex associates with roX1 and roX2 RNAs transcribed from the X chromosome. (A) Schematic outline of the RIP method. N1, N3 and N6 correspond to native nuclear extracts sonicated for 1, 3 and 6 min, respectively. FA corresponds to formaldehyde-cross-linked nuclear extract. (B) The tiling array results are computed as the ratio between the RIP value and the value of the corresponding nuclear input RNA preparation; MSL2-RIPs (blue), MOF-RIPs (green). Ratios calculated between the RIP values and a MOCK-RIP (instead of input) yield comparable results (data not shown). The plots show the mean enrichment ratios obtained using a bandwidth of 90 bp. Numbers on the x-axis denote chromo- somal position along the X chromosome in kilobase. The y-axis shows the RIP enrichment as the log2-ratio. In the resulting proﬁle, enrichments >1 (which correspond approximately to the top 1% of binding) are shown. Genes expressed from left to right are shown above the horizontal line and genes expressed in the opposite direction are shown below the line. The high peaks within the yellow boxes are roX1 and roX2, respectively. Below the enrichment plots, the MRE sites are indicated (orange) as previously characterized by (13). No signiﬁcant correlation of immunoprecipitated RNAs to MREs was found. the X chromosome and the autosomes are comparable (Supplementary Figure S1). In the formaldehyde ﬁxed samples (FA), X chromosome transcripts in general were enriched compared with transcript on other chromosomes. We therefore compared our RIP-chip data with available ChIP-chip data representing the MSL-complex bind- ing proﬁle. The results showed that the RIP from the native nuclear extract is highly enriched in roX RNAs while the RIP experiment from the formaldehyde cross-linked nuclear extract is in addition enriched in transcripts from genes targeted by the MSL complex (Figure 2A–C). This is probably a consequence of cross-linked chromatin fragments including RNA moieties linked by active transcription. A detailed analysis of the roX1- and roX2-precipitated RNAs shows that the entire roX1 and roX2 RNAs are immunoprecipitated by antibodies against both MOF and MSL2 (Figure 2D–E and Supplementary Figure S2). A higher enrichment of roX2 compared to roX1 was observed in all conditions except in the formaldehyde cross-linked sample, suggesting that roX2 is more stably bound to the complex in S2 cells. The MSL complex is strongly associated with nuclear msl2 mRNA It is well established that the roX genes can exert their activity in trans as well as in cis (24). We, therefore, asked whether any autosomal RNA has a stable associ- ation with the MSL complex. Visual inspection of enrich- ment curves readily identiﬁed a second target with a comparable enrichment ratio to roX1 and roX2, namely the msl2 transcript itself (Figure 3A). A ranking of all nuclear transcripts according to their mean enrichment ratio showed that msl2 binding is indeed comparable with the roX enrichments. Furthermore, msl1 also scored highly in this ranking, although much lower than roX1, roX2 and msl2 (Figure 3B and Supplementary Figure S3). It should be stressed that not only the enrichment ratio but also the absolute amount of msl2 RNA pulled down with the MSL complex are comparable to roX levels