protocol. After washing in ChIP washing buffer (UPSTATE), the beads and antibody complexes were resuspended in 1.5 ml of 3C ligation mix, containing 3200 U of T4 ligase, overnight at 15uC. After ligation, the samples were purified as in the 3C protocol. After 40 cycles of PCR, bands were visualised on an agarose gel and compared to the band obtained with 3C. RNAi depletion and cell-cycle synchronization RNAi knockdown of cohesin was done as described previously [6]. To obtain cells enriched in G2 and G1 phase HB2 cells were synchronised by double thymidine block: addition of 3 mM thymidine for 16 h, removal of thymidine by washing with PBS and release of the cells from the block for 8 h, addition of 3 mM thymidine for another 16 h for the second block. The cells are released from the second block by washing with PBS and cells are harvested after 6 h for enrichment in G2 phase and after 14 hours for enrichment in G1 phase. The enrichment of the cells in the respective cell cycle phases was controlled by FACS. To obtain cells enriched in G1 and G2 phase and depleted of the cohesin subunit SCC1 the siRNA transfection was performed either 6 hours before starting the first thymidine block (for G2 phase) or 2 hours after releasing the cells from the first thymidine block (for G1 phase). The siRNA oligos (sense-GGU- GAAAAUGGCAUUACGGtt and antisense CCGUAAUGC- CAUUUUCACCtt, Ambion) were annealed according to man- ufacturer’s instruction and used at a final concentration of 75 nM. The siRNA transfection was performed using lipofectamine RNAiMAX (Invitrogen). Statistical analysis of 3C after RNAi Two-way ANOVA with Bonferroni’s post-test was performed using GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. Normalised values of 3 biological replicate experiments for each ligation combination in a given anchor set was analysed by two- way ANOVA, with RNAi/control being one set of factors and restriction sites being the other set of factors. A Bonferrroni post post-test enabled comparison of multiple replicates at each restriction site. The Bonferroni correction lowers the P value considered significant to 0.05 divided by the number of comparisons. Thus in n rows of data with two columns (Control and RNAi), the P value has to be less than 0.05/n, for any particular row in order to be considered significant with P,0.05. This correction ensures that the 5% probability applies to the entire set of comparisons, and not separately to each individual comparison. RNA and DNA FISH To obtain a probe for RNA FISH two PCR products of 2000 bp and 600 bp (primer sequences available upon request) were generated from the last exon of the human IGF2 gene, mixed and labeled with dig-11-dUTP using the Biotin High Prime Kit (Roche). For the DNA probe the human BAC RP11-650021 spanning the IGF2 gene (chr.11: pos. 2057305–2245714) was directly labeled with Alexa 594 by random priming. Cells on coverslips were fixed for 15 min with 4% formaldehyde, 5% acetic acid in PBS and stored after another PBS wash in 70% ethanol at 4uC. Denaturation and hybridisation of the slides and probes was done as described in [54]. The biotin labelled probe was detected by successive incubations with mouse-anti biotin (DAKO, 1:500 dilution) and FITC-conjugated goat-anti-mouse antibodies (Jack- son ImmunoResearch Laboratories Inc., 1:500 dilution) and after dehydration mounted using Vectashield with DAPI (Vector Cohesin Mediates Chromatin Conformation Laboratories). The slides were analysed on a Leica DMRBE microscope equipped with a Hamatsu CCD (C4880) camera with a 100X objective. Adobe Photoshop was used to colour the images and generate the overlay figures. Supporting Information Figure S1 Schematic representation of BglII restriction sites at the IGF2-H19 locus. (A) INS, IGF2 and the H19 gene are displayed together with the cohesin/CTCF binding regions. Blue boxes represent genes and the green oval represents the downstream enhancer (Enh). Red rectangles indicate the positions of the CTCF/ cohesin binding regions CTCF AD, CCD, ICR, and CTCF DS. Vertical blue lines in a row show BglII restriction sites across the whole locus. Sites that were analysed for ligation (3C RS) are labelled with numbers. The third line shows the position of specific regions analysed for 3C and these are enlarged in the panels below. Note that CTCF AD has a restriction site 3 Kb far from DMR0 and can be analysed separately from other sites in the IGF2 locus. In panels B-E BglII restriction sites are depicted as red vertical lines relative to BamHI restriction sites shown as black lines and labelled with letters as in Figure 1. Black arrows and grey arrowheads indicate positions of anchor- and 3C- primers respectively. (B) Enlarged region encompassing the CTCF AD, the DMR0 and the IGF2 gene showing exons as boxes (blue for coding- and white for non-coding exons). Promoters are shown as arrows above the exons. The DMR0 region is shown as a yellow bar. (C) Enlargement of the intervening region between IGF2 and H19 showing the position of the CCD (red box) relative to the BglII and BamHI restriction sites. (D) Enlargement of ICR and H19 region. The CTCF/cohesin binding region is shown as red box and the position of the ICR is marked with a yellow box. (E) Enlargement of the enhancer and the CTCF DS region (green oval and red box respectively). Found at: doi:10.1371/journal.pgen.1000739.s001 (0.66 MB TIF) Figure S2 Chromatin conformation of the IGF2-H19 locus using BglII restriction. (A) Schematic representation of a 350 Kb genomic region including INS and the IGF2-H19 locus. Blue boxes represent the position of genes and the green oval depicts the localisation of the enhancer (Enh). Red boxes below represent the CTCF/cohesin binding regions CTCF AD, CCD, ICR, and CTCF DS. Positions of BglII restriction sites are marked with vertical blue lines and sites analysed by 3C are showed with numbers. Vertical pink lines in panel (B–D) indicate the position of the two different BglII restriction sites that were used as anchors. In each panel the respective anchor is marked with a thicker pink line. The X-axis is labelled according to the genomic position and position 0 was arbitrarily fixed 42 Kb upstream of INS. In (B,C) unsynchronised cells were analysed for the 3C interactions of the locus. (B) Associations detected with the anchor site in the ICR (restriction site 22 as anchor). The ICR associates with the CTCF AD and also with P2. (C) Associations with the CTCF AD (restriction site 4 as anchor). Interactions can be detected with the CCD and CTCF DS. (D) CTCF AD association frequencies (restriction site 4 as anchor) in cohesin depleted (SCC1 RNAi) and control (G2 control) cells synchronised in G2 phase. Interactions can be detected with the CCD and CTCF DS. Associations with the CCD were significantly decreased after SCC1 RNAi while interactions with the CTCF DS are only mildly affected. These results are similar to that obtained in Figure 7 with BamHI enzyme. Found at: doi:10.1371/journal.pgen.1000739.s002 (0.64 MB TIF) Figure S3 Chromatin conformation of the IGF2-H19 locus using an alternative anchor site in the ICR. (A) Schematic representation PLoS Genetics | www.plosgenetics.org 13 November 2009 | Volume 5 | Issue 11 | e1000739