specificity was determined by Western blot in CBP RNAi-treated Drosophila S2 cells (Figure S1). The rabbit antibody was further compared to an affinity-purified guinea-pig anti-dCBP aa 1–178 serum [9]. ChIP experiments show that the two CBP antibodies precipitate DNA in a quantitatively similar manner (Figure S1). A guinea-pig anti-Dorsal serum (Su3) was provided by Christos Samakovlis (Stockholm University). The following antibodies recognizing histone modifications were used: H3 (ab1791), H3K9ac (ab4441), H3K9me3 (ab8898), H3K18ac (ab1191), H3K27ac (ab4729), H3K27me3 (ab6002), and IgG (ab6722) were from Abcam, and H4Ac (Upstate 06-598) was from Millipore. Drosophila embryo collections Two- to four-hour old Drosophila embryos were collected on grape-juice plates, dechorionated, and used to prepare chromatin extracts as described below or fixed for in situ hybridization. Wild- type embryos were collected from w1118 flies, and embryos where Dorsal fails to enter nuclei were collected from gd7 homozygous mothers. Embryos with uniformly high levels of Dorsal in all nuclei were collected from Toll10B heterozygous females obtained directly from the balanced stock (Toll10B/TM3 Sb Ser/OR60). Tollrm9/ Tollrm10 trans-heterozygous females were used to collect embryos with intermediate levels of Dorsal in all nuclei. In situ hybridization Whole-mount RNA in situ hybridization using digoxigenin- labeled probes was performed as described previously [43,44]. Co-immunoprecipitation Two to four hour old embryos were dechorionated and crushed in a Dounce homogeniser in Lysis buffer (10 mM Tris pH 8, 140 mM NaCl, 1.5 mM MgCl2, 1% NP40, and proteinase inhibitors (Roche)). Lysate was shaken at 4uC and centrifuged. Pre-clearing and immunoprecipitation with the CBP antibody was done as previously described [45], except that Protein A Dynabeads (Invitrogen) were used. The immunoprecipitate was separated on 12% SDS-PAGE, transferred to nitrocellulose membrane, and probed with the Dorsal antibody diluted 1:1000. ChIP Two to four hour embryos were dechorionated, crushed in a Dounce homogeniser, and cross-linked with 1.8% formaldehyde in Buffer A1 (60 mM KCl, 15 mM NaCl, 15 mM Hepes pH 7.9, 4 mM MgCl2, 0.5 M DTT, 0.5% Triton X100, supplemented with proteinase inhibitor tablets, Roche) for 15 minutes at room temperature. The reaction was stopped with 0.225 M glycine and nuclei washed 3 times in Buffer A1 and once in Lysis buffer (140 mM NaCl, 15 mM Hepes pH 7.9, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 1% Triton X100, 0.5 M DTT, supplemented with proteinase inhibitor tablets, Roche). Nuclei were resuspended in Lysis buffer with 0.1% SDS and 0.5% N-lauroylsarcosine and sonicated in a Bioruptor (Diagenode). Chromatin extract was centrifuged to remove debris and diluted in an equal amount of lysis buffer, followed by snap freezing in liquid nitrogen and stored at 280uC. A mix of Protein A and G Dynabeads (Invitrogen) blocked with 1 mg/ml BSA (Sigma Aldrich) were mixed with indicated antibodies. A bead-antibody complex was formed at 4uC for at least 4 hours. Beads with bound antibody were captured on magnet, and beads were resuspended in chromatin extract corresponding to 30–40 ml of embryos followed by incubation at 4uC over night. Beads were washed 5 minutes each with sonication buffer (50 mM Hepes, 140 mM NaCl, 1 mM EDTA, CBP Occupancy in Drosophila Embryos 1% Triton, 0.1% sodium deoxycholate, 0.1% SDS), WashA (as sonication buffer, but with 500 mM NaCl), WashB (20 mM Tris pH 8, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and TE. Beads were transferred in TE to new tubes and resuspended in Elution buffer (50 mM Tris pH 8, 50 mM NaCl, 2 mM EDTA, 0.75% SDS, 20 mg/ml RNase A, 20 mg/ml glycogen). Cross-linking was reversed at 68uC for at least 4 hours and Proteinase K treated followed by DNA purification with phenol-chloroform extraction and ethanol precipitation. The DNA was resuspended in 200 ml 0.16TE. The ChIP material was analysed either by qPCR or sent to the Uppsala Genome Center for SOLiD (TM) ChIP-Seq Library preparation (Rev date 18 March 2010), size selection (100– 150 bp+adapters 90 bp <250 bp), and sequencing using SOLiD4 50 bp fragment run. Approximately 10 ChIPs for each genotype were pooled and used for the ChIP-seq libraries. For ChIP-qPCR, duplicates with 2 ml DNA each were used for analysis by qPCR, using 300 nM primers (Table S8), and iQ SYBR green supermix, run on a CFX96 Real-Time system from BioRad. Average of the two duplicates were compared to input, and then normalized to the Mi-2 locus or to two intergenic sites with background levels of binding. For histone antibodies, values were further normalized to total amount of histone H3. Processing of ChIP–seq data Reads were aligned against the Drosophila melanogaster reference sequence (release 5) using the classical mapping in Applied Biosystems Bioscope software v1.2.1. The number of uniquely mapping reads was 21,667,438 (wild-type input), 17,131,635 (wild type CBP), 25,683,953 (gd7 input) and 18,325,130 (gd7 CBP). Average read-count per nucleotide was calculated for IP and input samples. For regions where the IP sample had at least the average read-count, a ratio of IP-input (in log2 scale) was calculated. If the read count in the input was below the average read-count (in the input sample) it was set to the average. All ratio values were then adjusted by reducing each value with the average read-count in IP minus the average read-count in input. This linear adjustment was to normalize for differences in sequencing depth of IP and input. Then the ratio value at an interval of 35 bp was extracted across the genome and median smoothed using a window size of 350 bp. Windows with fewer than 5 data points were discarded. The dynamic range of CBP in wild type was 22.0 to +5.1 and CBP in gd7 21.8 to +5.1. To calculate peaks and bound regions, the 5% highest ratio values in both wild-type and gd7 were extracted, corresponding to a cut-off of 1.9 in wild type and 1.9 in gd7. Bound regions were then defined as regions of at least 200 bp and a region was extended as long as there was a value within 200 bp of the previous value. The value of each detected region was set to the average of the highest five consecutive ratio values. The center of the peak was set to the middle position of the five highest consecutive ratio values. When comparing CBP in gd7 to CBP in wild type we did not normalize the two data sets to each other since the dynamic range and the 5% cut-off was more or less identical. Calculating overlap between data sets When the overlap of bound regions of dataset X was to be compared to bound regions of other datasets first all regions of dataset X was used. Overlaps of at least one nucleotide were scored. Then an increasing cut-off for the binding values of data set X was applied. We used 20 cut-offs from the lowest to the highest binding value of dataset X in steps of maximum value minus minimum value then divided by 20. We plotted percent PLoS Genetics | www.plosgenetics.org 13 June 2012 | Volume 8 | Issue 6 | e1002769